Methods for producing a protein using an avian lysozyme promoter

ABSTRACT

The invention includes methods of producing proteins in transgenic avians containing nucleic acids in their genome which contain an exogenous lysozyme gene expression controlling nucleotide sequence which typically is linked to a polynucleotide encoding a heterologous polypeptide.

This application is a continuation of U.S. patent application Ser. No. 11/699,257, filed Jan. 26, 2007, which is a continuation-in-part of U.S. patent application Ser. No. 10/114,739, filed Apr. 1, 2002, which claims the benefit from provisional application Ser. No. 60/351,550 filed Jan. 25, 2002 and is a continuation-in-part of U.S. patent application Ser. No. 09/922,549, filed Aug. 3, 2001, which claims the benefit of provisional application Ser. No. 60/280,004, filed Mar. 30, 2001. The disclosure of U.S. patent application Ser. Nos. 11/699,257; 10/114,739 and 09/922,549 are incorporated herein in their entirety by reference.

FIELD OF THE INVENTION

The present invention relates generally to the use of avian lysozyme gene expression control or controlling regions, for example, from the chicken. More specifically, the invention relates to recombinant nucleic acids and expression vectors, transfected cells and transgenic animals, in particular transgenic avians such as transgenic chickens, that contain an avian lysozyme gene expression controlling regions operably linked to a polypeptide-encoding nucleic acid and, optionally, a chicken lysozyme 3′ domain. The present invention also relates to the expression of a polypeptide-encoding nucleic acid under the control of an exogenous avian lysozyme gene expression controlling region.

BACKGROUND

The field of transgenics was initially developed to understand the action of a single gene in the context of the whole animal and the phenomena of gene activation, expression and interaction. Transgenics technology has also been used to produce models for various diseases in humans and other animals and is among the most powerful tools available for the study of genetics, and the understanding of genetic mechanisms and function. From an economic perspective, the use of transgenic technology to convert animals into “protein factories” for the production of specific proteins or other substances of pharmaceutical interest (Gordon et al., 1987, Biotechnology 5: 1183-1187; Wilmut et al., 1990, Theriogenology 33: 113-123) offers significant advantages over more conventional methods of protein production by gene expression.

Heterologous nucleic acids have been engineered so that an expressed protein may be joined to a protein or peptide that will allow secretion of the transgenic expression product into milk or urine, from which the protein may then be recovered. These procedures have had limited success and may require lactating animals, with the attendant costs of maintaining individual animals or herds of large species, including cows, sheep, or goats.

Historically, transgenic animals have been produced almost exclusively by microinjection of the fertilized egg. The pronuclei of fertilized eggs are microinjected in vitro with foreign, i.e., xenogeneic or allogeneic, heterologous DNA or hybrid DNA molecules. The microinjected fertilized eggs are then transferred to the genital tract of a pseudopregnant female (e.g., Krimpenfort et al., in U.S. Pat. No. 5,175,384).

One system that holds potential for expressing foreign proteins is the avian reproductive system. The production of an avian egg begins with formation of a large yolk in the ovary of the hen. The unfertilized oocyte or ovum is positioned on top of the yolk sac. After ovulation, the ovum passes into the infundibulum of the oviduct where it is fertilized, if sperm are present, and then moves into the magnum of the oviduct, which is lined with tubular gland cells. These cells secrete the egg-white proteins, including ovalbumin, lysozyme, ovomucoid, conalbumin and ovomucin, into the lumen of the magnum where they are deposited onto the avian embryo and yolk.

Advantages of using the hen oviduct as a protein bioreactor include the high levels of protein production, the promise of proper folding and post-translation modification of the target protein, the ease of product recovery, and the shorter developmental period of birds such as chickens compared to other animal species. As a result, efforts have been made to create transgenic chickens, for example, by expressing heterologous proteins in the oviduct by means of microinjection of DNA. See, for example, PCT Publication WO 97/47739, the disclosure of which is incorporated in its entirety herein by reference.

The chicken lysozyme gene is highly expressed in the myeloid lineage of hematopoietic cells, and in the tubular glands of the mature hen oviduct. See, for example, Hauser et al., 1981, Hematol. and Blood Transfusion 26: 175-178; Schutz et al., 1978, Cold Spring Harbor Symp. Quart. Biol. 42: 617-624 (the disclosures of which are incorporated in their entirety herein by reference). In one embodiment, elements of the regulatory region of the lysozyme locus can extend over at least 12 kb of DNA 5′ upstream of the transcription start site and can comprise a number of elements that have been individually isolated and characterized. Known elements include three enhancer sequences at about −6.1 kb, −3.9 kb, and −2.7 kb (Grewal et al., 1992, Mol. Cell. Biol. 12: 2339-2350; Banifer et al., 1996, J. Mol. Med. 74: 663-671), a hormone responsive element (Hecht et al., 1988, E.M.B.O. J. 7: 2063-2073), a silencer element and a complex proximal promoter. The constituent elements of the lysozyme gene expression control region are identifiable as DNAase 1 hypersensitive chromatin sites (DHS). They may be differentially exposed to nuclease digestion depending upon the differentiation stage of the cell. For example, in the multipotent progenitor stage of myelomoncytic cell development, or in erythroblasts, the silencer element is a DHS. At the myeloblast stage, a transcription enhancer located −6.1 kb upstream from the gene transcription start site is a DHS, while at the later monocytic stage another enhancer, at −2.7 kb becomes DNAase sensitive (Huber et al., 1995, DNA and Cell Biol. 14: 397-402).

Scattered throughout the chicken genome, including the chicken lysozyme locus, are short stretches of nucleic acid that resemble features of Long Terminal Repeats (LTRs) of retrovirus. The function of these elements is unclear but most likely help define the DHS regions of a gene locus (Stein et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 6485-6489).

Flanking the lysozyme gene, including the regulatory region, are matrix attachment regions (5′ MAR and 3′ MAR), alternatively referred to as “scaffold attachment regions” or SARs. The outer boundaries of the chicken lysozyme locus have been defined by the MARs (Phi-Van et al., 1988, E.M.B.O. J. 7: 655-664; Phi-Van, L. and Stratling, W. H., 1996, Biochem. 35: 10735-10742). Deletion of a 1.32 kb or a 1.45 kb halves region, each comprising half of a 5′ MAR, reduces positional variation in the level of transgene expression (Phi-Van and Stratling, supra).

The 5′ matrix-associated region (5′ MAR), located about −11.7 kb upstream of the chicken lysozyme transcription start site, can increase the level of gene expression by limiting the positional effects exerted against a transgene (Phi-Van et al., 1988, supra). At least one other MAR is located 3′ downstream of the protein encoding region. Although MAR nucleic acid sequences are conserved, little cross-hybridization is seen, indicating significant overall sequence variation. However, MARs of different species can interact with the nucleomatrices of heterologous species, to the extent that the chicken lysozyme MAR can associate with the plant tobacco nucleomatrix as well as that of the chicken oviduct cells (Mlynarona et al., 1994, Cell 6: 417-426; von Kries et al., 1990, Nucleic Acids Res. 18: 3881-3885).

Gene expression must be considered not only from the perspective of cis-regulatory elements associated with a gene, and their interactions with trans-acting elements, but also with regard to the genetic environment in which they are located. Chromosomal positioning effects (CPEs), therefore, are the variations in levels of transgene expression associated with different locations of the transgene within the recipient genome. An important factor governing CPE upon the level of transgene expression is the chromatin structure around a transgene, and how it cooperates with the cis-regulatory elements. The cis-elements of the lysozyme locus are confined within a single chromatin domain (Bonifer et al., 1996, supra; Sippel et al., pgs. 133-147 in Eckstein F. & Lilley D. M. J. (eds), “Nucleic Acids and Molecular Biology”, Vol. 3, 1989, Springer.

Deletion of a cis-regulatory element from a transgenic lysozyme locus is sufficient to reduce or eliminate positional independence of the level of gene expression (Banifer et al., 1996, supra). There is also evidence indicating that positional independence conferred on a transgene requires the cotransfer of many kilobases of DNA other than just the protein encoding region and the immediate cis-regulatory elements.

The lysozyme promoter region of chicken is active when transfected into mouse fibroblast cells and linked to a reporter gene such as the bacterial chloramphenicol acetyltransferase (CAT) gene. The promoter element is also effective when transiently transfected into chicken promacrophage cells. In each case, however, the presence of a 5′ MAR element increased positional independency of the level of transcription (Stief et al., 1989, Nature 341: 343-345; Sippel et al., pgs. 257-265 in Houdeline L. M. (ed), “Transgenic Animals: Generation and Use”).

The ability to direct the insertion of a transgene into a site in the genome of an animal where the positional effect is limited offers predictability of results during the development of a desired transgenic animal, and increased yields of the expressed product. Sippel and Steif disclose, in U.S. Pat. No. 5,731,178, methods to increase the expression of genes introduced into eukaryotic cells by flanking a transcription unit with scaffold attachment elements, in particular the 5′ MAR isolated from the chicken lysozyme gene. The transcription unit disclosed by Sippel and Steif was an artificial construct that combined only the −6.1 kb enhancer element and the proximal promoter element (base position −579 to +15) from the lysozyme gene. Other promoter associated elements were not included. However, although individual cis-regulatory elements have been isolated and sequenced, together with short regions flanking DNA, the entire nucleic acid sequence comprising the functional 5′ upstream region of the lysozyme gene has not been determined in its entirety and therefore not employed as a functional promoter to allow expression of a heterologous transgene.

What is needed are efficient transcription promoters that will allow expression of a transgene in avian cells, in particular, in the oviduct cells (e.g., tubular gland cells) of a transgenic avian.

SUMMARY OF THE INVENTION

Briefly described, the present invention relates to nucleic acids comprising avian lysozyme gene expression controlling regions which may be isolated. In one particularly useful aspect, the invention provides for transgenic avians, and methods of there production, wherein the transgenic avians contain in their genome an exogenous nucleotide sequence comprising an avian lysozyme gene expression controlling region. Typically, the exogenous avian lysozyme gene expression controlling region is operably linked to an exogenous or heterologous coding sequence (e.g., a coding sequence which encodes an exogenous or heterologous peptide or protein). In one useful embodiment, the exogenous coding sequence is expressed in an oviduct cell of the transgenic avian, for example, in a tubular gland cell of the transgenic avian. Typically, in accordance with the invention, the expressed product is secreted from the oviduct cell, for example, secreted from the oviduct cell (e.g., tubular gland cell) into the oviduct.

Examples of transgenic avians that can be produced in accordance with the present invention include, without limitation, transgenic chickens, transgenic turkeys, transgenic ducks and transgenic quail. The transgenic avian may be a chimeric transgenic avian. That is, some but not all, of the cells of the transgenic avian may contain an exogenous nucleotide sequence which includes a recombinant lysozyme gene expression controlling region. Such chimeric avians can be germ-line chimerics in which some of the avian's germ cells contain the exogenous nucleotide sequence. Such germ-line chimeric avians can give rise to transgenic avians in which essentially all the cells of the avians contain the exogenous nucleotide sequence containing a recombinant lysozyme gene expression controlling region, as is understood in the art of animal breeding and avian transgenesis. See, for example, US patent publication No. 2006/0015960, filed Jun. 24, 2005, the disclosure of which is incorporated in its entirety herein by reference.

In one particularly useful embodiment, the lysozyme gene expression controlling region is linked to an exogenous nucleotide sequence such as a nucleotide sequence encoding a therapeutic protein, e.g., a human protein (i.e., a protein normally produced in a human). In accordance with the invention, the transgenic avian can produce the exogenous protein, for example, in the oviduct cells of the transgenic avian. In such instance the exogenous protein is typically deposited in egg white produced by the transgenic avian. In one particularly useful aspect, the transgenic avian can produce an egg such as a hard shelled egg containing the exogenous protein.

The invention specifically contemplates the application of any useful avian lysozyme gene expression controlling region encompassed in SEQ ID NO: 67 for producing transgenic avians of the invention. For example, the invention contemplates the use of nucleotide sequence corresponding to any fragment or portion of SEQ ID NO: 67 having gene expression controlling activity (e.g., promoter activity), for the production of transgenic avians as disclosed herein. Also contemplated is the use of nucleotide sequences that can function as a gene expression controlling region (e.g., function as a promoter) in which the nucleotide sequences are at least about 75% identical to the nucleotide sequence depicted in SEQ ID NO: 67. In addition, it is contemplated that such nucleotide sequences can be at least about 80%, and at least about 85%, and at least about 90%, and at least about 91%, and at least about 92%, and at least about 93%, and at least about 94%, and at least about 95%, and at least about 96%, and at least about 97%, and at least about 98%, and at least about 99% identical to the nucleotide sequence depicted in SEQ ID NO: 67. Also contemplated is the use of nucleotide sequences that can function as a gene expression controlling region (e.g., function as a promoter) in which the nucleotide sequences are at least about 15% identical to a fragment of the nucleotide sequence depicted in SEQ ID NO: 67. In addition, it is contemplated that nucleotide sequences that can function as a gene expression controlling region can be at least about 80%, and at least about 85%, and at least about 90%, and at least about 91%, and at least about 92%, and at least about 93%, and at least about 94%, and at least about 95%, and at least about 96%, and at least about 97%, and at least about 98%, and at least about 99% identical to a fragment of the nucleotide sequence depicted in SEQ ID NO: 67.

Examples of specifically contemplated sequences for the production of transgenic avians (including chimeras, e.g., germ-line chimeras and their progeny birds) such as transgenic chickens, transgenic quail and transgenic turkeys include, a nucleotide sequence at least 80% identical to nucleotides 7665 to 11863 of SEQ ID NO: 67 and a nucleotide sequence at least 90% identical to nucleotides 7665 to 11863 of SEQ ID NO: 67 and a nucleotide sequence at least 95% identical to nucleotides 7665 to 11863 of SEQ ID NO: 67 and a nucleotide sequence at least 96% identical to nucleotides 7665 to 11863 of SEQ ID NO: 67 and a nucleotide sequence at least 97% identical to nucleotides 7665 to 11863 of SEQ ID NO: 67 and a nucleotide sequence at least 98% identical to nucleotides 7665 to 11863 of SEQ ID NO: 67 and a nucleotide sequence at least 99% identical to nucleotides 7665 to 11863 of SEQ ID NO: 67 and a nucleotide sequence identical to nucleotides 7665 to 11863 of SEQ ID NO: 67; a nucleotide sequence at least 80% identical to nucleotides 5381 to 11863 of SEQ ID NO: 67 and a nucleotide sequence at least 90% identical to nucleotides 5381 to 11863 of SEQ ID NO: 67 and a nucleotide sequence at least 95% identical to nucleotides 5381 to 11863 of SEQ ID NO: 67 and a nucleotide sequence at least 96% identical to nucleotides 5381 to 11863 of SEQ ID NO: 67 and a nucleotide sequence at least 97% identical to nucleotides 5381 to 11863 of SEQ ID NO: 67 and a nucleotide sequence at least 98% identical to nucleotides 5381 to 11863 of SEQ ID NO: 67 and a nucleotide sequence at least 99% identical to nucleotides 5381 to 11863 of SEQ ID NO: 67 and a nucleotide sequence identical to nucleotides 5381 to 11863 of SEQ ID NO: 67; a nucleotide sequence at least 80% identical to nucleotides 9159 to 11863 of SEQ ID NO: 67 and a nucleotide sequence at least 90% identical to nucleotides 9159 to 11863 of SEQ ID NO: 67 and a nucleotide sequence at least 95% identical to nucleotides 9159 to 11863 of SEQ ID NO: 67 and a nucleotide sequence at least 96% identical to nucleotides 9159 to 11863 of SEQ ID NO: 67 and a nucleotide sequence at least 97% identical to nucleotides 9159 to 11863 of SEQ ID NO: 67 and a nucleotide sequence at least 98% identical to nucleotides 9159 to 11863 of SEQ ID NO: 67 and a nucleotide sequence at least 99% identical to nucleotides 9159 to 11863 of SEQ ID NO: 67 and a nucleotide sequence identical to nucleotides 9159 to 11863 of SEQ ID NO: 67.

Often, the exogenous nucleotide sequence which contains the recombinant lysozyme gene expression controlling region also includes a vector. That is, the nucleotide sequence which is incorporated into the genome of the transgenic avian (i.e., the transgene) can include a vector. The invention contemplates any useful vector being part of the transgene including, without limitation, a plasmid vector, a viral vector and an artificial chromosome.

In one embodiment, an avian lysozyme gene expression controlling region of the invention, such as one which is encompassed in SEQ ID NO: 67 as described herein, is employed in a SIN vector to produce a transgenic avian (e.g., transgenic chicken) in accordance with the invention. In another embodiment, an avian lysozyme gene expression controlling region of the invention such as one encompassed in SEQ ID NO: 67 as describe above is employed in a retroviral vector which does not contain an antibiotic resistance marker gene to produce a transgenic avian (e.g., transgenic chicken) in accordance with the invention. In another embodiment, an avian lysozyme gene expression controlling region of the invention such as one encompassed in SEQ ID NO: 67 as describe above is employed in a SIN vector which does not contain an antibiotic resistance marker gene (i.e., does not contain a promoter for an antibiotic resistance marker) to produce a transgenic avian (e.g., transgenic chicken) in accordance with the invention.

In one embodiment, an isolated nucleic acid of the present invention is useful for reducing the chromosomal positional effect of a transgene operably linked to the lysozyme gene expression control region and transfected into a recipient cell. By isolating a region of the avian genome extending from 5′ upstream of a 5′ MAR of the lysozyme locus to the junction between the signal peptide sequence and a polypeptide-encoding region, cis-elements are also included to allow gene expression in a tissue-specific manner. The lysozyme promoter region of the present invention, therefore, will allow expression of an operably linked heterologous nucleic acid insert in a transfected avian cell such as, for example, an oviduct cell.

One aspect of the present invention provides a novel isolated nucleic acid that is located immediately 5′ upstream of the native lysozyme-encoding region of the chicken lysozyme gene locus. The novel isolated avian nucleic acid sequence encoding a lysozyme gene expression control region comprises at least one 5′ matrix attachment region, an intrinsically curved DNA region, at least one transcription enhancer element, a negative regulatory element, at least one hormone responsive element, at least one avian CR1 repeat element, and a proximal lysozyme promoter and signal peptide-encoding region. Interspersed between these constituent elements are stretches of nucleic acid that serve at least to organize the above elements in an ordered or functional array relative to a polypeptide-encoding region.

In one embodiment of the present invention the gene expression controlling region, e.g., promoter of the invention is isolated from a chicken.

The promoter of the present invention may be operably linked with a selected nucleic acid insert, wherein the nucleic acid insert encodes a polypeptide desired to be expressed in a transfected cell. The nucleic acid insert may be placed in frame with a signal peptide sequence. Translation initiation may start with the signal peptide and continue through the nucleic acid insert, thereby producing an expressed polypeptide having the desired amino acid sequence.

The sequence of the expressed nucleic acid insert may be optimized for codon usage by a host cell. This may be determined from the codon usage of at least one, and preferably more than one, proteins expressed in a chicken cell. For example, the codon usage may be determined from the nucleic acid sequences encoding the proteins ovalbumin, lysozyme, ovomucin and ovotransferrin of chicken.

The recombinant DNA of the present invention may further comprise a polyadenylation signal sequence that will allow the transcript directed by the novel lysozyme gene expression control region to proceed beyond the nucleic acid insert encoding a polypeptide and allow the transcript to further comprise a 3′ untranslated region and a polyadenylated tail. Any functional polyadenylation signal sequence may be linked to the 3′ end of the nucleic acid insert including the SV40 polyadenylation signal sequence, bovine growth hormone adenylation sequence or the like.

The recombinant DNA of the invention may comprise the chicken lysozyme 3′ domain operably linked to the nucleic acid insert encoding a polypeptide. The 3′ domain may include a 3′ untranslated region, a polyadenylation signal and a 3′ MAR that may reduce positional variation in transgenic avians.

Yet another aspect of the present invention is expression vectors suitable for delivery to a recipient cell or animal for expression of the therein. The expression vector of the present invention may comprise an isolated avian lysozyme gene expression control region operably linked to a nucleic acid insert encoding a polypeptide, and optionally a polyadenylation signal sequence. The expression vector may further comprise a bacterial plasmid sequence, a viral nucleic acid sequence, or fragments or variants thereof that may allow for replication of the vector in a suitable host.

Another aspect of the present invention is a method of expressing a heterologous polypeptide in a eukaryotic cell by transfecting the cell with a recombinant DNA comprising an avian lysozyme gene expression controlling region operably linked to a nucleic acid insert encoding a polypeptide and, optionally, a polyadenylation signal sequence, and culturing the transfected cell in a medium suitable for expression of the heterologous polypeptide under the control of the avian lysozyme gene expression control region.

Also within the scope of the present invention are recombinant cells, tissues and animals containing non-naturally occurring recombinant nucleic acid molecules according to the present invention and described above. In one embodiment of the present invention, the transformed cell is a chicken oviduct cell and the nucleic acid insert comprises the chicken lysozyme gene expression control region, a nucleic acid insert encoding a human interferon α2 and codon optimized for expression in an avian cell, and an SV40 polyadenylation sequence.

Another aspect of the invention provides for a vector comprising a first and second coding sequence and a promoter in operational and positional relationship to the first and second coding sequence to express the first and second coding sequence in an avian oviduct. In this aspect, the vector may include an internal ribosome entry site (IRES) element positioned between the first and second coding sequence, wherein the first coding sequence codes for protein X and the second coding sequence codes for protein Y, and wherein one or both of protein X and protein Y are deposited into the egg (e.g., egg white) of a hard shell egg.

For example, protein X may be a light chain (LC) of a monoclonal antibody and protein Y may be a heavy chain (HC) of a monoclonal antibody. Alternatively, the protein encoded by the second coding sequence (e.g., enzyme) may be capable of providing post-translational modification of the protein encoded by the first coding sequence. The vector optionally includes additional coding sequences and additional IRES elements, such that each coding sequence in the vector is separated from another coding sequence by an IRES element. Other examples of employing an IRES which are contemplated for use in the present invention are disclosed in, for example, U.S. patent application Ser. No. 11/047,184, filed Jan. 31, 2005, the disclosure of which is incorporated in its entirety herein by reference.

The invention also contemplates methods of producing an avian egg which contains proteins such as therapeutic or pharmaceutical proteins including monoclonal antibodies, enzymes and other proteins. Such methods may include providing a vector with a promoter, coding sequences, and at least one IRES element; creating transgenic cells or tissue by introducing the vector into avian embryonic blastodermal cells, wherein the vector sequence is randomly inserted into the avian genome; and deriving a mature transgenic avian from the transgenic cells or tissue. The transgenic avian so derived may express the coding sequences in its oviduct, and the resulting protein secreted into the oviduct lumen, so that the protein is deposited into the egg white of a hard shell egg. In addition, the invention includes progeny of the transgenic avians which produce eggs containing the recombinant protein. Typically, the progeny will either contain the transgene in essentially all the cells of the bird or none of the cells of the progeny bird will contain the transgene.

One important aspect of the present invention relates to avian hard shell eggs (e.g., chicken hard shell eggs) which contain an exogenous peptide or protein including, but not limited to, a pharmaceutical protein. The exogenous peptide or protein may be encoded by a transgene of a transgenic avian. In one embodiment, the exogenous peptide or protein (e.g., pharmaceutical protein) is glycosylated. The protein may be present in any useful amount. In one embodiment, the protein is present in an amount in a range of between about 0.1 μg per hard-shell egg and about 1 gram per hard-shell egg. In another embodiment, the protein is present in an amount in a range of between about 1 μg per hard-shell egg and about 1 gram per hard-shell egg. For example, the protein may be present in an amount in a range of between about 10 μg per hard-shell egg and about 1 gram per hard-shell egg (e.g., a range of between about 10 μg per hard-shell egg and about 400 milligrams per hard-shell egg). In one embodiment, the protein is present in an amount in a range of between about 500 μg per hard-shell egg and about 50 milligrams per hard-shell egg.

In one embodiment, the exogenous protein, for example, the exogenous pharmaceutical protein, is present in the egg white of the egg. In one embodiment, the protein is present in an amount in a range of between about 1 ng per milliliter of egg white and about 0.2 gram per milliliter of egg white. For example, the protein may be present in an amount in a range of between about 0.1 μg per milliliter of egg white and about 0.2 gram per milliliter of egg white (e.g., the protein may be present in an amount in a range of between about 1 μg per milliliter of egg white and about 100 milligrams per milliliter of egg white. In one embodiment, the protein is present in an amount in a range of between about 1 μg per milliliter of egg white and about 50 milligrams per milliliter of egg white. For example, the protein may be present in an amount in a range of about 1 μg per milliliter of egg white and about 10 milligrams per milliliter of egg white (e.g., the protein may be present in an amount in a range of between about 1 μg per milliliter of egg white and about 5 milligrams per milliliter of egg white). In one embodiment, the protein is present in an amount in a range of about 50 μg per milliliter of egg white and about 5 milligrams per milliliter of egg white.

The invention contemplates the production of hard shell eggs containing any useful protein including one or more pharmaceutical proteins. Such proteins include, but are not limited to, hormones, immunoglobulins or portions of immunoglobulins, cytokines (e.g., GM-CSF, G-CSF, erythropoietin and interferon) and CTLA4. The invention also includes the production of hard shell eggs containing fusion proteins including, but not limited to, immunoglobulins or portions of immunoglobulins fused to certain useful peptide sequences. In one embodiment, the invention provides for the production of hard shell eggs containing an antibody Fc fragment. For example, the eggs may contain an Fc-CTLA4 fusion protein.

The avians developed from the blastodermal cells into which the vector has been introduced are the G0 generation and are referred to as “founders”. Founder birds are typically chimeric for each inserted transgene. That is, only some of the cells of the G0 transgenic bird contain the transgene(s). The G0 generation typically is also hemizygous for the transgene(s). The G0 generation may be bred to non-transgenic animals to give rise to G1 transgenic offspring which are also hemizygous for the transgene and contain the transgene(s) in essentially all of the bird's cells. The G1 hemizygous offspring may be bred to non-transgenic animals giving rise to G2 hemizygous offspring or may be bred together to give rise to G2 offspring homozygous for the transgene. Substantially all of the cells of birds which are positive for the transgene that are derived from G1 offspring will contain the transgene(s). In one embodiment, hemizygotic G2 offspring from the same line can be bred to produce G3 offspring homozygous for the transgene. In one embodiment, hemizygous G0 animals are bred together to give rise to homozygous G1 offspring containing two copies of the transgene(s) in each cell of the animal. These are merely examples of certain useful breeding schemes and the present invention contemplates the employment of any useful breeding scheme such as those known to individuals of ordinary skill in the art.

Any useful combination of features described herein is included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art

Additional objects and aspects of the present invention will become more apparent upon review of the detailed description set forth below when taken in conjunction with the accompanying figures, which are briefly described as follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 a, FIG. 1 b, FIG. 1 c and FIG. 1 d illustrate the primers (SEQ ID NOS: 1-64) used in the sequencing of the lysozyme gene expression controlling region of SEQ ID NO: 67.

FIG. 2 schematically illustrates the approximately 12 kb lysozyme gene expression controlling region shown in SEQ ID NO: 67) indicating the relative positions and orientations of the primers (SEQ ID NOS: 1-64) used in the sequencing thereof.

FIG. 3 a, FIG. 3 b, FIG. 3 c and FIG. 3 d illustrate the nucleic acid sequence (SEQ ID NO: 65) comprising the chicken lysozyme gene expression controlling region shown in SEQ ID NO: 67, the nucleic acid sequence of SEQ ID NO: 66 encoding the chicken expression optimized human interferon α2b (IFNMAGMAX) which is underlined in the figures and the SV40 polyadenylation signal sequence shown in SEQ ID NO: 68, which is in bold print in the figures.

FIG. 4 illustrates the nucleic acid sequence of SEQ ID NO: 66 encoding the chicken expression optimized human interferon α2b (IFNMAGMAX).

FIG. 5 a, FIG. 5 b, FIG. 5 c and FIG. 5 d illustrate the nucleic acid sequence of SEQ ID NO: 67 encoding the chicken lysozyme gene expression controlling region.

FIG. 6 illustrates the nucleic acid sequence of SEQ ID NO: 68 encoding the SV40 polyadenylation signal sequence.

FIG. 7 a, FIG. 7 b and FIG. 7 c illustrate a portion of the nucleic acid sequence of SEQ ID NO: 69 encoding the chicken lysozyme 3′ domain.

FIG. 8 a, FIG. 8 b, FIG. 8 c, FIG. 8 d, FIG. 8 e, FIG. 8 f, FIG. 8 g, FIG. 8 h, FIG. 8 i and FIG. 8 j illustrate the nucleic acid sequence of SEQ ID NO: 74 encoding the lysozyme gene expression controlling region shown in SEQ ID NO: 67 linked to the nucleic acid insert encoding the chicken expression-optimized human interferon α2b (IFNMAGMAX) (SEQ ID NO: 66) which is underlined in the figure and is in turn linked to the chicken lysozyme 3′ domain (SEQ ID NO: 69) which is shown in bold print. A fragment of a pBluescript cloning vector approximately 44 nucleotides in length is present between the IFNMAGMAX and the lysozyme 3′ domain and is shown in lower case (FIG. 8 f). The nucleotide sequence shown in lower case 3′ of the chicken lysozyme 3′ domain is nucleotide sequence from the cloning vectors pPolyIII and pBluescript (See, FIG. 8 i to 8 j).

FIG. 9 illustrates the yield of the human interferon α2b, optimized for chicken expression (IFNMAGMAX), in transfected quail oviduct cultured cells.

FIG. 10 illustrates the yield of the human interferon α2b, optimized for chicken expression (IFNMAGMAX), in chicken myelomonocytic HD 11 cells transfected with plasmids pAVIJCR-A115.93.1.2, pAVIJC-A212.89.2.3 or pAVIJCR-A212.89.2.1.

FIG. 11 illustrates the expression of α2b human interferon in the blood of transgenic chickens #8305 and #AA61, as compared to standards.

FIG. 12 illustrates the gel analysis of PCR products derived from the serum of transgenic birds. Lane and Samples applied to the gel were: 1, marker; 2, 8301; 3, 8303; 4, 8305 5, 8305; 6, 8307; 7, 8309; 8, 8311; 9, marker; 10, 8313; 11, 8305; 12, 8305; 13, Neg. Ctrl; 14, Pos. Ctrl (500 pg)+Neg. Ctrl; 15, Pos. Ctrl (500 pg).

FIG. 13 illustrates a self-inactivating vector of the invention containing a 4.2 kb lysozyme promoter fragment operably linked to an interferon alpha 2 coding sequence and signal peptide coding sequence. The 5′ long terminal repeat (LTR) of the vector is the complete LTR of an RSV virus. The 3′ LTR has a deletion in the enhancer such that when the retroviral region integrates the 5′ LTR is inactivated. The nucleotide sequence of the vector of FIG. 13 is shown in SEQ ID NO: 75.

FIG. 14 is a bar graph illustrating expression levels of IFNa in the egg white of a transgenic quail. G0 quail was produced by injection of pALV-SIN-4.0-Lys-IFNa-2B retroviral vector transduction particles into Japanese quail embryos.

DETAILED DESCRIPTION

Reference now will be made in detail to certain embodiments of the invention, one or more examples of which are illustrated in the accompanying drawings. Each example is provided by way of explanation of the invention, not limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications, combinations, additions, deletions and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment can be used in another embodiment to yield a still further embodiment. It is intended that the present invention covers such modifications, combinations, additions, deletions and variations as come within the scope of the appended claims and their equivalents.

This description uses gene nomenclature accepted by the Cucurbit Genetics Cooperative as it appears in the Cucurbit Genetics Cooperative Report 18:85 (1995), herein incorporated by reference in its entirety. Using this gene nomenclature, genes are symbolized by italicized Roman letters. If a mutant gene is recessive to the normal type, then the symbol and name of the mutant gene appear in italicized lower case letters.

For convenience, certain terms employed in the specification, examples, and appended claims are collected here.

DEFINITIONS

The term “animal” is used herein to include all vertebrate animals, including avians and may include humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages.

The term “avian” as used herein refers to any species, subspecies or race of organism of the taxonomic class ava, such as, but not limited to, such organisms as chicken, turkey, duck, goose, quail, pheasants, parrots, finches, hawks, crows and ratites including ostrich, emu and cassowary. The term includes the various known strains of Gallus gallus, or chickens, (for example, White Leghorn, Brown Leghorn, Barred-Rock, Sussex, New Hampshire, Rhode Island, Ausstralorp, Minorca, Amrox, California Gray, Italian Partidge-colored), as well as strains of turkeys, pheasants, quails, duck, ostriches and other poultry commonly bred in commercial quantities.

The phrase “based on” or “derived from” as in a retroviral vector being based on or derived from a particular retrovirus or based on a nucleotide sequence of a particular retrovirus mean that the genome of the retroviral vector contains a substantial portion of the nucleotide sequence of the genome of the particular retrovirus. The substantial portion may be a particular gene or nucleotide sequence such as the nucleotide sequence encoding the gag, pol and/or env proteins or other structural or functional nucleotide sequence of the virus genome such as sequences encoding the LTRs or may be substantially the complete retrovirus genome, for example, most (e.g., more than 60% or more than 70% or more than 80% or more than 90%) or all of the retrovirus genome, as will be apparent from the context in the specification as the knowledge of one skilled in the art. Examples of retroviral vectors that are based on or derived from a retrovirus are the NL retroviral vectors (e.g., NLB) which are based on the ALV retrovirus as disclosed in Cosset et al, Journal of Virology (1991) vol 65, p 3388-3394.

The terms “heterologous”, “exogenous” and “foreign” are used interchangeably herein and in general refer to a biomolecule such as a nucleic acid or a protein that is not normally found in a certain cell, tissue or other component contained in or produced by an organism. For example, a protein that is heterologous or exogenous to an egg is a protein that is not normally found in the egg.

As used herein, the terms “heterologous”, “exogenous” and “foreign” with reference to nucleic acids, such as DNA and RNA, are used interchangeably and refer to nucleic acid that does not occur naturally as part of a chromosome, a genome or cell in which it is present or which is found in a location(s) and/or in amounts that differ from the location(s) and/or amounts in which it occurs in nature. It can be nucleic acid that is not endogenous to the genome, chromosome or cell and has been exogenously introduced into the genome, chromosome or cell. Examples of heterologous DNA include, but are not limited to, a DNA comprising a gene expression control region and DNA that encodes a product or products, for example, RNA or protein product. Examples of heterologous DNA include, but are not limited to, lysozyme gene expression control regions of the invention once isolated from the avian and as used thereafter, e.g., after introduction into an avian genome.

The term “nucleic acid” as used herein refers to any natural and synthetic linear and sequential arrays of nucleotides and nucleosides, for example cDNA, genomic DNA, mRNA, tRNA, oligonucleotides, oligonucleosides and derivatives thereof. For ease of discussion, such nucleic acids may be collectively referred to herein as “constructs,” “plasmids,” or “vectors.” Representative examples of the nucleic acids of the present invention include bacterial plasmid vectors including expression, cloning, cosmid and transformation vectors such as, but not limited to, pBR322, animal viral vectors such as, but not limited to, modified adenovirus, influenza virus, polio virus, pox virus, retroviruses such as avian leukosis virus (ALV) retroviral vector, a murine leukemia virus (MLV) retroviral vector, and a lentivirus vector, and the like. In addition, the vector may be a nucleic acid sequence which includes an LTR of an avian leukosis virus (ALV) retroviral vector, a murine leukemia virus (MLV) retroviral vector, or a lentivirus vector. NL vectors such as NLB, NLD and NLA are also contemplated for use in methods of the present invention. Vectors may be derived from bacteriophage nucleic acid, and synthetic oligonucleotides like chemically synthesized DNA or RNA. The term “nucleic acid” further includes modified or derivatised nucleotides and nucleosides such as, but not limited to, halogenated nucleotides such as, but not only, 5-bromouracil, and derivatised nucleotides such as biotin-labeled nucleotides.

The term “isolated nucleic acid” as used herein refers to a nucleic acid with a structure (a) not identical to that of any naturally occurring nucleic acid or (b) not identical to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes, and includes DNA, RNA, or derivatives or variants thereof. The term covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic molecule but is not flanked by at least one of the coding sequences that flank that part of the molecule in the genome of the species in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic nucleic acid of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any vector or naturally occurring genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), ligase chain reaction (LCR) or chemical synthesis, or a restriction fragment; (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein, and (e) a recombinant nucleotide sequence that is part of a hybrid sequence that is not naturally occurring. Isolated nucleic acid molecules of the present invention can include, for example, natural allelic variants as well as nucleic acid molecules modified by nucleotide deletions, insertions, inversions, or substitutions such that the resulting nucleic acid molecule still essentially encodes a lysozyme gene expression control region or a variant thereof of the present invention.

By the use of the term “enriched” in reference to nucleic acid it is meant that the specific DNA or RNA sequence constitutes a significantly higher fraction of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. Enriched does not imply that there are no other DNA or RNA sequences present, just that the relative amount of the sequence of interest has been significantly increased. The other DNA may, for example, be derived from a yeast or bacterial genome, or a cloning vector, such as a plasmid or a viral vector. The term “significant” as used herein is used to indicate that the level of increase is useful to the person making such an increase.

It is advantageous for some purposes that a nucleotide sequence is in purified form. The term “purified” in reference to nucleic acid represents that the sequence has increased purity relative to the natural environment.

The terms “polynucleotide,” “oligonucleotide,” and “nucleic acid sequence” are used interchangeably herein and include, but are not limited to, coding sequences (polynucleotide(s) or nucleic acid sequence(s) which are transcribed and translated into polypeptide in vitro or in vivo when placed under the control of appropriate regulatory or control sequences); control sequences (e.g., translational start and stop codons, promoter sequences, ribosome binding sites, polyadenylation signals, transcription factor binding sites, transcription termination sequences, upstream and downstream regulatory domains, enhancers, silencers, and the like); and regulatory sequences (DNA sequences to which a transcription factor(s) binds and alters the activity of a gene's promoter either positively (induction) or negatively (repression)). No limitation as to length or to synthetic origin are suggested by the terms described herein.

As used herein the terms “polypeptide” and “protein” refer to a polymer of amino acids of three or more amino acids in a serial array, linked through peptide bonds. The term “polypeptide” includes proteins, protein fragments, protein analogues, oligopeptides and the like. The term “polypeptides” contemplates polypeptides as defined above that are encoded by nucleic acids, produced through recombinant technology (isolated from an appropriate source such as a bird), or synthesized. The term “polypeptides” further contemplates polypeptides as defined above that include chemically modified amino acids or amino acids covalently or noncovalently linked to labeling ligands.

The term “fragment” as used herein to refer to a nucleic acid (e.g., cDNA) refers to an isolated portion of the subject nucleic acid constructed artificially (e.g., by chemical synthesis) or by cleaving a natural product into multiple pieces, using restriction endonucleases or mechanical shearing, or a portion of a nucleic acid synthesized by PCR, DNA polymerase or any other polymerizing technique well known in the art, or expressed in a host cell by recombinant nucleic acid technology well known to one of skill in the art. The term “fragment” as used herein may also refer to an isolated portion of a polypeptide, wherein the portion of the polypeptide is cleaved from a naturally occurring polypeptide by proteolytic cleavage by at least one protease, or is a portion of the naturally occurring polypeptide synthesized by chemical methods well known to one of skill in the art.

The term coding sequence as used herein refers to nucleotide sequences or nucleic acid sequences (including both RNA or DNA) that encode genetic information for the synthesis of a whole RNA, a whole protein, or any portion of such whole RNA or whole protein. Nucleotide sequences that are not naturally part of a particular organism's genome are referred to as “foreign nucleotide sequences,” “heterologous nucleotide sequences” or “exogenous nucleotide sequences”. “Heterologous products” are RNAs or proteins encoded by “foreign, heterologous or exogenous nucleotide sequences” and are, therefore, not naturally expressed in the cell. A nucleotide sequence that has been isolated and then reintroduced into the same type (e.g., same species) of organism is not considered to be a naturally occurring part of a particular organism's genome and is therefore considered exogenous or heterologous.

The term “expressed” or “expression” as used herein refers to the transcription from a gene to give an RNA nucleic acid molecule at least complementary in part to a region of one of the two nucleic acid strands of the gene. The term “expressed” or “expression” as used herein also refers to the translation from said RNA nucleic acid molecule to give a protein, a polypeptide or a portion thereof.

As used herein, the term “locus” or “loci” refers to the site of a gene on a chromosome. Pairs of genes control hereditary traits, each in the same position on a pair of chromosomes. These gene pairs, or alleles, may both be dominant or both be recessive in expression of that trait. In either case, the individual is said to be homozygous for the trait controlled by that gene pair. If the gene pair (alleles) consists of one dominant and one recessive trait, the individual is heterozygous for the trait controlled by the gene pair. Natural variation in genes or nucleic acid molecules caused by, for example, recombination events or resulting from mutation, gives rise to allelic variants with similar, but not identical, nucleotide sequences. Such allelic variants typically encode proteins with similar activity to that of the protein encoded by the gene to which they are compared, because natural selection typically selects against variations that alter function. Allelic variants can also comprise alterations in the untranslated regions of the gene as, for example, in the 3′ or 5′ untranslated regions or can involve alternate splicing of a nascent transcript, resulting in alternative exons being positioned adjacently.

The term “operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.

The terms “transcription regulatory sequences” and “gene expression control regions” and “gene expression controlling regions” as used herein refer to nucleotide sequences that are associated with a nucleic acid sequence and which regulate the transcriptional expression of a coding sequence. Exemplary transcription regulatory sequences include enhancer elements, hormone response elements, steroid response elements, negative regulatory elements, and the like. The “transcription regulatory sequences” may be isolated and incorporated into a vector nucleic acid to enable regulated transcription in appropriate cells of portions of the vector DNA. The “transcription regulatory sequence” may precede, but is not limited to, the region of a nucleic acid sequence that is in the region 5′ of the end of a protein coding sequence that may be transcribed into mRNA. Transcriptional regulatory sequences may also be located within a protein coding region, in regions of a gene that are identified as “intron” regions, or may be in regions of nucleic acid sequence that are in the region of nucleic acid.

The term “promoter” as used herein refers to the DNA sequence that determines the site of transcription initiation from an RNA polymerase. A “promoter-proximal element” may be a regulatory sequence within about 200 base pairs of the transcription start site.

The terms “matrix attachment regions” or “SAR elements” as used herein refer to DNA sequences having an affinity or intrinsic binding ability for the nuclear scaffold or matrix. The MAR elements of the chicken lysozyme locus were described by Phi-Van et al., 1988, E.M.B.O. J. 76: 665-664 and Phi-Van L. and Stratling, W. H., 1996, Biochem. 35: 10735-10742, the contents of which are incorporated herein by reference in their entireties.

The term “coding region” as used herein refers to a continuous linear arrangement of nucleotides which may be translated into a protein. A full length coding region is translated into a full length protein; that is, a complete protein as would be translated in its natural state absent any post-translational modifications. A full length coding region may also include any leader protein sequence or any other region of the protein that may be excised naturally from the translated protein.

The term “complementary” as used herein refers to two nucleic acid molecules that can form specific interactions with one another. In the specific interactions, an adenine base within one strand of a nucleic acid can form two hydrogen bonds with thymine within a second nucleic acid strand when the two nucleic acid strands are in opposing polarities. Also in the specific interactions, a guanine base within one strand of a nucleic acid can form three hydrogen bonds with cytosine within a second nucleic acid strand when the two nucleic acid strands are in opposing polarities. Complementary nucleic acids as referred to herein, may further comprise modified bases wherein a modified adenine may form hydrogen bonds with a thymine or modified thymine, and a modified cytosine may form hydrogen bonds with a guanine or a modified guanine.

The term “probe” as used herein, when referring to a nucleic acid, refers to a nucleotide sequence that can be used to hybridize with and thereby identify the presence of a complementary sequence, or a complementary sequence differing from the probe sequence but not to a degree that prevents hybridization under the hybridization stringency conditions used. The probe may be modified with labels such as, but not only, radioactive groups, biotin, and the like that are well known in the art.

The term “capable of hybridizing under stringent conditions” as used herein refers to annealing a first nucleic acid to a second nucleic acid under stringent conditions as defined below. Stringent hybridization conditions typically permit the hybridization of nucleic acid molecules having at least 70% nucleic acid sequence identity with the nucleic acid molecule being used as a probe in the hybridization reaction. For example, the first nucleic acid may be a test sample or probe, and the second nucleic acid may be the sense or antisense strand of a lysozyme gene expression control region or a fragment thereof. Hybridization of the first and second nucleic acids may be conducted under stringent conditions, e.g., high temperature and/or low salt content that tend to disfavor hybridization of dissimilar nucleotide sequences. Alternatively, hybridization of the first and second nucleic acid may be conducted under reduced stringency conditions, e.g., low temperature and/or high salt content that tend to favor hybridization of dissimilar nucleotide sequences. Low stringency hybridization conditions may be followed by high stringency conditions or intermediate medium stringency conditions to increase the selectivity of the binding of the first and second nucleic acids. The hybridization conditions may further include reagents such as, but not limited to, dimethyl sulfoxide (DMSO) or formamide to disfavor still further the hybridization of dissimilar nucleotide sequences. A suitable hybridization protocol may, for example, involve hybridization in 6×SSC (wherein 1×SSC comprises 0.015 M sodium citrate and 0.15 M sodium chloride), at 65° C. in an aqueous solution, followed by washing with 1×SSC at 65° C. Formulae to calculate appropriate hybridization and wash conditions to achieve hybridization permitting 30% or less mismatch between two nucleic acid molecules are disclosed, for example, in Meinkoth et al., 1984, Anal. Biochem. 138: 267-284; the content of which is herein incorporated by reference in its entirety. Protocols for hybridization techniques are well known to those of skill in the art and standard molecular biology manuals may be consulted to select a suitable hybridization protocol without undue experimentation. See, for example, Sambrook et al. 1989, “Molecular Cloning: A Laboratory Manual”, 2nd ed., Cold Spring Harbor Press, the contents of which are herein incorporated by reference in its entirety.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) from about pH 7.0 to about pH 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C.

The terms “unique nucleic acid region” and “unique protein (polypeptide) region” as used herein refer to sequences present in a nucleic acid or protein (polypeptide) respectively that is not present in any other nucleic acid or protein sequence. The terms “conserved nucleic acid region” as referred to herein is a nucleotide sequence present in two or more nucleic acid sequences, to which a particular nucleic acid sequence can hybridize under low, medium or high stringency conditions. The greater the degree of conservation between the conserved regions of two or more nucleic acid sequences, the higher the hybridization stringency that will allow hybridization between the conserved region and a particular nucleic acid sequence.

The terms “percent sequence identity” or “percent sequence similarity” as used herein refer to the degree of sequence identity between two nucleic acid sequences or two amino acid sequences as determined using the algorithm of Karlin and Attschul, 1990, Proc. Natl. Acad. Sci. 87: 2264-2268, modified as in Karlin and Attschul, 1993, Proc. Natl. Acad. Sci. 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Attschul et al., 1990, T. Mol. Biol. Q15: 403-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to a reference polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Attschul et al., 1997, Nucl. Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g. XBLAST and NBLAST) are used. See ncbi.nlm.nih.gov. Other algorithms, programs and default settings may also be suitable such as, but not only, the GCG-Sequence Analysis Package of the U.K. Human Genome Mapping Project Resource Centre that includes programs for nucleotide or amino acid sequence comparisons.

The term “sense strand” as used herein refers to a single stranded DNA molecule from a genomic DNA that may be transcribed into RNA and translated into the natural polypeptide product of the gene. The term “antisense strand” as used herein refers to the single strand DNA molecule of a genomic DNA that is complementary with the sense strand of the gene.

The term “antisense DNA” as used herein refers to a gene sequence DNA that has a nucleotide sequence complementary to the “sense strand” of a gene when read in reverse orientation, i.e., DNA read into RNA in a 3′ to 5′ direction rather than in the 5′ to 3′ direction. The term “antisense RNA” is used to mean an RNA nucleotide sequence (for example that encoded by an antisense DNA or synthesized complementary with the antisense DNA). Antisense RNA is capable of hybridizing under stringent conditions with an antisense DNA. The antisense RNA of the invention is useful for regulating expression of a “target gene” either at the transcriptional or translational level. For example, transcription of the subject nucleic acids may produce antisense transcripts that are capable of inhibiting transcription by inhibiting initiation of transcription or by competing for limiting transcription factors; the antisense transcripts may inhibit transport of the “target RNA”, or, the antisense transcripts may inhibit translation of “target RNA”.

The term “nucleic acid vector” as used herein refers to a natural or synthetic single or double stranded plasmid or viral nucleic acid molecule that can be transfected or transformed into cells and replicate independently of, or within, the host cell genome. A circular double stranded plasmid can be linearized by treatment with an appropriate restriction enzyme based on the nucleotide sequence of the plasmid vector. A nucleic acid can be inserted into a vector by cutting the vector with restriction enzymes and ligating the pieces together. The nucleic acid molecule can be RNA or DNA.

The term “expression vector” as used herein refers to a nucleic acid vector that comprises the lysozyme gene expression control region operably linked to a nucleotide sequence coding at least one polypeptide. As used herein, the term “regulatory sequences” includes promoters, enhancers, and other elements that may control gene expression. Standard molecular biology textbooks such as Sambrook et al. eds., 1989, “Molecular Cloning: A Laboratory Manual”, 2nd ed., Cold Spring Harbor Press may be consulted to design suitable expression vectors that may further include an origin of replication and selectable gene markers. It should be recognized, however, that the choice of a suitable expression vector and the combination of functional elements therein depends upon multiple factors including the choice of the host cell to be transformed and/or the type of protein to be expressed.

The terms “transformation” and “transfection” as used herein refer to the process of inserting a nucleic acid into a host. Many techniques are well known to those skilled in the art to facilitate transformation or transfection of a nucleic acid into a prokaryotic or eukaryotic organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt such as, but not only a calcium or magnesium salt, an electric field, detergent, or liposome mediated transfection, to render the host cell competent for the uptake of the nucleic acid molecules, and by such methods as sperm-mediated and restriction-mediated integration.

The term “transfecting agent” as used herein refers to a composition of matter added to the genetic material for enhancing the uptake of heterologous DNA segment(s) into a eukaryotic cell, preferably an avian cell, and more preferably a chicken male germ cell. The enhancement is measured relative to the uptake in the absence of the transfecting agent. Examples of transfecting agents include adenovirus-transferrin-polylysine-DNA complexes. These complexes generally augment the uptake of DNA into the cell and reduce its breakdown during its passage through the cytoplasm to the nucleus of the cell. These complexes can be targeted to the male germ cells using specific ligands that are recognized by receptors on the cell surface of the germ cell, such as the c-kit ligand or modifications thereof.

Other preferred transfecting agents include but are not limited to lipofectin, lipfectamine, DIMRIE C, Supeffect, and Effectin (Qiagen), unifectin, maxifectin, DOTMA, DOGS (Transfectam; dioctadecylamidoglycylsp-ermine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOTAP (1,2-dioleoyl-3-trimethylammonium propane), DDAB (dimethyl dioctadecytammonium bromide), DHDEAB (N,N-di-n-hexadecyl-N,N-dihydroxyeth-yl ammonium bromide), HDEAB (N-n-hexadecylN,N-dihydroxyethylammonium bromide), polybrene, or poly(ethylenimine) (PEI). These non-viral agents have the advantage that they can facilitate stable integration of xenogeneic DNA sequences into the vertebrate genome, without size restrictions commonly associated with virus-derived transfecting agents.

The term “recombinant cell” refers to a cell that has a new combination of nucleic acid segments that are not covalently linked to each other in nature. A new combination of nucleic acid segments can be introduced into an organism using a wide array of nucleic acid manipulation techniques available to those skilled in the art. A recombinant cell can be a single eukaryotic cell, or a single prokaryotic cell, or a mammalian cell. The recombinant cell may harbor a vector that is extragenomic. An extragenomic nucleic acid vector does not insert into the cell's genome. A recombinant cell may further harbor a vector or a portion thereof that is intragenomic. The term intragenomic defines a nucleic acid construct incorporated within the recombinant cell's genome.

The terms “recombinant nucleic acid” and “recombinant DNA” as used herein refer to combinations of at least two nucleic acid sequences that are not naturally found in a eukaryotic or prokaryotic cell. The nucleic acid sequences may include, but are not limited to, nucleic acid vectors, gene expression regulatory elements, origins of replication, suitable gene sequences that when expressed confer antibiotic resistance, protein-encoding sequences and the like. The term “recombinant polypeptide” is meant to include a polypeptide produced by recombinant DNA techniques such that it is distinct from a naturally occurring polypeptide either in its location, purity or structure. Generally, such a recombinant polypeptide will be present in a cell in an amount different from that normally observed in nature.

As used herein, a “transgenic animal” is any animal, such as an avian species, including the chicken, in which one or more of the cells of the avian may contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art. The nucleic acid is introduced into an animal, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus. The term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule. This molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA. In the typical transgenic animal, the transgene causes cells to express a recombinant form of the subject polypeptide, e.g., either agonistic or antagonistic forms, or in which the gene has been disrupted. The terms “chimeric animal” or “mosaic animal” are used herein to refer to animals in which a recombinant nucleotide sequence is found, or in which the recombinant nucleotide sequence is expressed in some but not all cells of the animal. The term “tissue-specific chimeric animal” indicates that the recombinant gene is present and/or expressed in some tissues but not others. A germ-line chimeric animal can give rise to a transgenic animal in which most or all cells

As used herein, the term “transgene” means a nucleic acid sequence (encoding, for example, a human protein) that is partly or entirely heterologous, i.e., foreign, to the transgenic animal or cell into which it is introduced, or, is homologous to an endogenous gene of the transgenic animal or cell into which it is introduced, but which is designed to be inserted, or is inserted, into the animal's genome in such a way as to alter the genome of the cell into which it is inserted (e.g., it is inserted at a location which differs from that of the natural gene or its insertion results in a knockout). A transgene according to the present invention will include one or more transcriptional regulatory sequences, polyadenylation signal sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.

The term “chromosomal positional effect (CPE)” as used herein refers to the variation in the degree of gene transcription as a function of the location of the transcribed locus within the cell genome. Random transgenesis may result in a transgene being inserted at different locations in the genome so that individual cells of a population of transgenic cells may each have at least one transgene, each at a different location and therefore each in a different genetic environment. Each cell, therefore, may express the transgene at a level specific for that particular cell and dependent upon the immediate genetic environment of the transgene. In a transgenic animal, as a consequence, different tissues may exhibit different levels of transgene expression.

Techniques useful for isolating and characterizing the nucleic acids and proteins of the present invention are well known to those of skill in the art and standard molecular biology and biochemical manuals may be consulted to select suitable protocols without undue experimentation. See, for example, Sambrook et al, 1989, “Molecular Cloning: A Laboratory Manual”, 2nd ed., Cold Spring Harbor, the content of which is herein incorporated by reference in its entirety.

Abbreviations:

Abbreviations used in the present specification include the following: aa, amino acid(s); bp, base pair(s); cDNA, DNA complementary to RNA; nt, nucleotide(s); SSC, sodium chloride-sodium citrate; DMSO, dimethyl sulfoxide; MAR; matrix attachment region.

Chicken lysozyme gene expression controlling region nucleic acid sequences: A series of PCR amplifications of template chicken genomic DNA were used to isolate the gene expression control region of the chicken lysozyme locus. Two amplification reactions used the PCR primer sets SEQ ID NOS: 1 and 2 and SEQ ID NOS: 3 and 4. The amplified PCR products were united as a contiguous isolated nucleic acid by a third PCR amplification step with the primers SEQ ID NOS: 1 and 4, as described in Example 1 below.

One isolated PCR-amplified product of the invention, comprising about 12 kb of the nucleic acid region 5′ upstream of the native chicken lysozyme gene locus, was cloned into the plasmid pCMV-LysSPIFNMM. pCMV-LysSPIFNMM comprises a modified nucleic acid insert encoding a human interferon α2 sequence and an SV40 polyadenylation signal sequence 3′ downstream of the interferon encoding nucleic acid. The sequence of SEQ ID NO: 66 of the nucleic acid insert encoding human interferon α2 was in accordance with avian cell codon usage, as determined from the nucleotide sequences encoding chicken ovomucin, ovalbumin, ovotransferrin and lysozyme. The novel chicken lysozyme gene expression control region, interferon-encoding insert and the SV40 polyadenylation signal sequence of the resulting plasmid construct pAVIJCR-A115.93.1.2, constructed as described in Example 1 below, was sequenced using the artificial oligonucleotide primers SEQ ID NOS: 1-64, as illustrated in FIGS. 1 and 2.

The nucleic acid sequence (SEQ ID NO: 65) (GenBank Accession No. AF405538) of the insert in pAVIJCR-A115.93.1.2 is shown in FIG. 3, with the modified human interferon α2 encoding nucleotide sequence of SEQ ID NO: 66 (GenBank Accession No. AF405539) and the novel chicken lysozyme gene expression control region SEQ ID NO: 67 (GenBank Accession No. AF405540) shown in FIGS. 4 and 5 respectively. A polyadenylation signal sequence that is suitable for operably linking to the polypeptide-encoding nucleic acid insert is the SV40 signal sequence of SEQ ID NO: 68, as shown in FIG. 6.

The plasmid pAVIJCR-A 115.93.1.2 was restriction digested with enzyme FseI to isolate a 15.4 kb DNA containing the lysozyme 5′ matrix attachment region (MAR) and the −12.0 kb lysozyme promoter during the expression of the interferon-encoding insert, as described in Example 2, below. Plasmid pIIIilys was restriction digested with MluI and XhoI to isolate an approximately 6 kb nucleic acids, comprising the 3′ lysozyme domain, the sequence of which (SEQ ID NO: 70) is shown in FIG. 7. The 15.4 kb and 6 kb nucleic acids were ligated and the 21.4 kb nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 70 (GenBank Assession No. AF 497473) as shown in FIG. 8 was transformed into recipient STBL4 cells as described in Example 2, below.

The inclusion of the novel isolated avian lysozyme gene expression control region of the present invention upstream of a codon-optimized interferon-encoding sequence in pAVIJCR-A115.93.1.2 allowed expression of the interferon polypeptide in transfected avian cells, as described in Example 5, below. The 3′ lysozyme domain shown in SEQ ID NO: 69, when operably linked downstream of the heterologous nucleic acid insert, also assists in providing for expression of the nucleic acid insert as described in Example 7, below. For example, the nucleic acid insert may encode a heterologous polypeptide such as the α2 interferon having sequence of SEQ ID NO: 66 (α2b). The invention also contemplates the use of nucleotide sequences which are at least about 75%, and at least about 80%, and at least about 85%, and at least about 90%, and at least about 91%, and at least about 92%, and at least about 93%, and at least about 94%, and at least about 95%, and at least about 96%, and at least about 97%, and at least about 98%, and at least about 99%, identical to the 3′ lysozyme domain such as the 3′ lysozyme domain shown in SEQ ID NO: 69. Functional fragments of SEQ ID NO: 66 are also encompassed by the invention.

It is further contemplated that any nucleic acid sequence encoding a polypeptide may be operably linked to the novel isolated avian lysozyme gene expression control region and optionally operably linked to the 3′ lysozyme domain SEQ ID NO: 69 so as to be expressed in a transfected avian cell. The plasmid construct pAVIJCR-A 115.93.1.2 was transfected into cultured quail oviduct cells, which were then incubated for about 72 hours. ELISA assays of the cultured media showed that the transfected cells synthesized a polypeptide detectable with anti-human interferon α2 antibodies. Plasmid construct pAVIJCR-A212.89.2.1 and pAVIJCR-A212.89.2.3 transfected into chicken myelomonocytic HD11 cells yield detectable human α2b interferon, as described in Example 9 and shown in FIG. 10, below.

One isolated chicken lysozyme gene expression control region of the present invention comprises the nucleotide elements that are positioned 5′ upstream of the lysozyme-encoding region of the native chicken lysozyme locus and which are necessary for the regulated expression of a downstream polypeptide-encoding nucleic acid. While not wishing to be bound by any one theory, the inclusion of at least one 5′ MAR element in the isolated control region may confer positional independence to a transfected gene operably linked to the novel lysozyme gene expression control region.

Isolated lysozyme gene expression control regions of the present invention can be useful for reducing the chromosomal positional effect of a transgene operably linked to the lysozyme gene expression control region and transfected into a recipient avian cell. By isolating a region of the avian genome extending from a point 5′ upstream of a 5′ MAR of the lysozyme locus to the junction between the signal peptide sequence and a polypeptide-encoding region, cis-regulatory elements are also included that may allow gene expression in a tissue-specific manner. The lysozyme promoter region of the present invention, therefore, will allow expression of an operably linked heterologous nucleic acid insert in a transfected avian cell such as, for example, an oviduct cell.

It is further contemplated that a recombinant DNA of the present invention may further comprise the chicken lysozyme 3′ domain (SEQ. ID NO: 69) linked downstream of the nucleic acid insert encoding a heterologous polypeptide. The lysozyme 3′ domain includes a nucleic acid sequence encoding a 3′ MAR domain that may cooperate with a 5′ MAR to direct the insertion of the construct of the present invention into the chromosome of a transgenic avian, or may act independently of the 5′ MAR.

One aspect of the present invention, therefore, provides a novel isolated nucleic acid that comprises the nucleotide sequence of SEQ ID NO: 67, shown in FIG. 5 and derivatives and variants thereof, that is located immediately 5′ upstream of the native lysozyme-encoding region of the chicken lysozyme gene locus.

In one embodiment of the novel isolated nucleic acid of the present invention, therefore, the avian nucleic acid sequence encoding a lysozyme gene expression control region comprises at least one 5′ matrix attachment region, an intrinsically curved DNA region, at least one transcription enhancer element, a negative regulatory element, at least one hormone responsive element, at least one avian CR1 repeat element, and a proximal lysozyme promoter and signal peptide-encoding region. Interspersed between these constituent elements are stretches of nucleic acid that serve at least to organize the above elements in an ordered array relative to a polypeptide-encoding region, such as that encoding for chicken lysozyme. It is contemplated to be within the scope of the present invention that the cis-elements of the lysozyme gene expression control region may be in any linear arrangement that can allow the formation of a transcript comprising the nucleotide sequence or its complement of a nucleic insert operably linked to the lysozyme gene expression control region.

In one embodiment of the present invention, the isolated nucleic acid may be isolated from an avian selected from the group consisting of a chicken, a turkey, a duck, a goose, a quail, a pheasant, a ratite, an ornamental bird or a feral bird.

In another embodiment of the present invention, the isolated nucleic acid is obtained from a chicken. In this embodiment, the isolated nucleic acid has the sequence of SEQ ID NO: 67, as shown in FIG. 5, or a functional fragment thereof. A functional fragment refers to a portion of SEQ ID NO: 67 which can function as a promoter in vivo.

Another aspect of the present invention provides a novel isolated nucleic acid that comprises the nucleic acid of SEQ ID NO: 69 encoding the chicken 3′ lysozyme domain operably liked to the nucleic acid having sequence of SEQ ID NO: 65.

One embodiment of the isolated nucleic acid of the present invention, therefore, is a lysozyme gene expression control region comprising the nucleic acid sequence of SEQ ID NO. 66 operably linked to a nucleic acid for expression in avian cells, and a chicken 3′ lysozyme domain having the nucleic acid sequence of SEQ ID NO: 70, as shown in FIG. 8.

In another embodiment of the isolated nucleic acid of the present invention, the nucleic acid for expression in avian cells encodes a therapeutic protein. In one embodiment, the coding sequence for the therapeutic protein is optimized for expression in avian cells.

Another aspect of the invention provides nucleic acids that can hybridize under high, medium or low stringency conditions to an isolated nucleic acid that encodes a chicken lysozyme gene expression control region having all, a derivative of, or a portion of the nucleic acid sequence of SEQ ID NO: 67 shown in FIG. 5. The nucleotide sequence determined from the isolation of the lysozyme gene expression control region from a chicken (SEQ ID NO: 67) will allow for the generation of probes designed for use in identifying homologs of lysozyme gene expression control regions in other avian species.

Fragments of a nucleic acid encoding a portion of the subject lysozyme gene expression control region are also within the scope of the invention. As used herein, a fragment of the nucleic acid encoding an active portion of a lysozyme gene expression control region refers to a nucleotide sequence having fewer nucleotides than the nucleotide sequence encoding the entire nucleic acid sequence of the lysozyme gene expression control region.

In one embodiment of the present invention, the nucleotide sequence of the isolated DNA molecule of the present invention may be used as a probe in nucleic acid hybridization assays for the detection of the lysozyme gene expression control region. The nucleotide sequence of the present invention may be used in any nucleic acid hybridization assay system known in the art, including, but not limited to, Southern blots, Southern, E. M., 1975, J. Mol. Biol. 98: 508, Northern blots, Thomas et al., 1980, Proc. Natl. Acad. Sci. 77: 5201-05, and Colony blots, Grunstein et al., 1975, Proc. Natl. Acad. Sci. 72: 3961-65, the disclosures of which are incorporated herein by reference in their entireties. Alternatively, the isolated DNA molecules of the present invention can be used in a gene amplification detection procedure such as a polymerase chain reaction, Erlich et al., 1991, Science 252: 1643-51, the disclosure of which is incorporated herein by reference in its entirety, or in restriction fragment length polymorphism (RFLP) diagnostic techniques, as described in pgs. 519-522 and 545-547 of Watson et al., 2nd ed., 1992, “Recombinant DNA”, Scientific American Books, the disclosure of which is incorporated herein by reference in its entirety.

Nucleotides constructed in accordance with the present invention can be labeled to provide a signal as a means of detection. For example, radioactive elements such as ³²P, ³H, and ³⁵S or the like provide sufficient half-life to be useful as radioactive labels. Other materials useful for labeling synthetic nucleotides include fluorescent compounds, enzymes and chemiluminescent moieties. Methods useful in selecting appropriate labels and binding protocols for binding the labels to the synthetic nucleotides are well known to those of skill in the art. Standard immunology manuals, such as Promega: Protocol and Applications Guide, 2nd Edition, 1991 Promega Corp., Madison, Wis., the disclosure of which is incorporated herein in its entirety, may be consulted to select an appropriate labeling protocol without undue experimentation.

In another embodiment of the present invention, an isolated nucleic acid molecule of the present invention includes a nucleic acid that is at least about 75%, and at least about 80%, and at least about 85%, and at least about 90%, and at least about 91%, and at least about 92%, and at least about 93%, and at least about 94%, and at least about 95%, and at least about 96%, and at least about 97%, and at least about 98%, and at least about 99%, identical to a chicken-derived lysozyme gene expression controlling region-encoding nucleic acid molecule as included in SEQ ID NO: 67.

In another embodiment of the present invention, an avian lysozyme gene expression control region gene or nucleic acid molecule can be an allelic variant of the gene expression controlling regions shown in SEQ ID NO: 67.

The present invention also contemplates the use of antisense nucleic acid molecules that are designed to be complementary to a coding strand of a nucleic acid (i.e., complementary to an mRNA sequence) or, alternatively, complimentary to a 5′ or 3′ untranslated region of the mRNA. Another use of synthetic nucleotides is as primers (DNA or RNA) for a polymerase chain reaction (PCR), ligase chain reaction (LCR), or the like.

Synthesized nucleotides can be produced in variable lengths. The number of bases synthesized will depend upon a variety of factors, including the desired use for the probes or primers. Additionally, sense or anti-sense nucleic acids or oligonucleotides can be chemically synthesized using modified nucleotides to increase the biological stability of the molecule or of the binding complex formed between the anti-sense and sense nucleic acids. For example, acridine substituted nucleotides can be synthesized. Protocols for designing isolated nucleotides, nucleotide probes, and/or nucleotide primers are well-known to those of ordinary skill, and can be purchased commercially from a variety of sources (e.g., Sigma Genosys, The Woodlands, Tex. or The Great American Gene Co., Ramona, Calif.).

The nucleic acid sequence of a chicken lysozyme gene expression control region nucleic acid molecule (e.g., included in SEQ ID NO: 67) of the present invention allows one skilled in the art to, for example, (a) make copies of those nucleic acid molecules by procedures such as, but not limited to, insertion into a cell for replication by the cell, by chemical synthesis or by procedures such as PCR or LCR, (b) obtain nucleic acid molecules which include at least a portion of such nucleic acid molecules, including full-length genes, full-length coding regions, regulatory control sequences, truncated coding regions and the like, (c) obtain lysozyme gene expression control region nucleic acid homologs in other avian species such as, but not limited to, turkey, duck, goose, quail, pheasant, parrot, finch, ratites including ostrich, emu and cassowary and, (d) to obtain isolated nucleic acids capable of hybridizing to an avian lysozyme gene expression control region nucleic acid and be used to detect the presence of nucleic acid-related sequences by complementation between the probe and the target nucleic acid.

Such nucleic acid homologs can be obtained in a variety of ways including by screening appropriate expression libraries with antibodies of the present invention, using traditional cloning techniques to screen appropriate libraries, amplifying appropriate libraries or DNA using oligonucleotide primers of the present invention in a polymerase chain reaction or other amplification method, and screening public and/or private databases containing genetic sequences using nucleic acid molecules of the present invention to identify targets. Examples of preferred libraries to screen, or from which to amplify nucleic acid molecules, include but are not limited to mammalian BAC libraries, genomic DNA libraries, and cDNA libraries. Similarly, preferred sequence databases useful for screening to identify sequences in other species homologous to chicken lysozyme gene expression control region include, but are not limited to, GenBank and the mammalian Gene Index database of The Institute of Genomics Research (TIGR).

Codon-Optimized Proteins

Another aspect of the present invention is a recombinant DNA molecule comprising the novel isolated avian lysozyme gene expression control region of the present invention operably linked to a selected polypeptide-encoding nucleic acid insert, and which may express the nucleic acid insert when transfected to a suitable host cell, preferably an avian cell. The nucleic acid insert may be placed in frame with a signal peptide sequence, whereby translation initiation from the transcript may start with the signal peptide and continue through the nucleic acid insert, thereby producing an expressed polypeptide having the desired amino acid sequence.

It is anticipated that the recombinant DNA, therefore, may further comprise a polyadenylation signal sequence that will allow the transcript directed by the novel lysozyme gene expression control region to proceed beyond the nucleic acid insert encoding a polypeptide and allow the transcript to further comprise a 3′ untranslated region and a polyadenylated tail. Any functional polyadenylation signal sequence may be linked to the 3′ end of the nucleic acid insert including the SV40 polyadenylation signal sequence, bovine growth hormone adenylation sequence or the like, or derivatives thereof.

In one embodiment of the recombinant DNA of the present invention, the polyadenylation signal sequence is derived from the SV40 virus.

In another embodiment of the recombinant DNA of the present invention, the polyadenylation signal has the nucleic acid sequence of SEQ ID NO: 68 or a variant thereof, as shown in FIG. 6.

It is further anticipated that the recombinant DNA of the present invention may further comprise the chicken lysozyme 3′ domain SEQ ID NO: 69, or a variant thereof. The lysozyme 3′ domain comprises a 3′ untranslated region, a polyadenylation sequence and at least on 3′ MAR.

Another aspect of the present invention is to provide nucleic acid sequences of a human interferon α2b protein optimized for expression in avian cells, and derivatives and fragments thereof.

In derivatives of proteins such as therapeutic proteins of the present invention, for example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid (i.e. conservative mutations) will not have a major effect on the biological activity of the resulting molecule. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids can be divided into four families: (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine, histidine; (3) nonpolar=alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar=glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids. In similar fashion, the amino acid repertoire can be grouped as (1) acidic=aspartate, glutamate; (2) basic=lysine, arginine histidine, (3) aliphatic=glycine, alanine, valine, leucine, isoleucine, serine, threonine, with serine and threonine optionally be grouped separately as aliphatic-hydroxyl; (4) aromatic=phenylalanine, tyrosine, tryptophan; (5) amide=asparagine, glutamine; and (6) sulfur-containing=cysteine and methionine. (see, for example, “Biochemistry”, 2nd ed, L. Stryer, ed., W H Freeman and Co., 1981). Peptides in which more than one replacement has taken place can readily be tested in the same manner.

One embodiment of the present invention is a recombinant DNA molecule comprising the isolated avian lysozyme gene expression control region of the present invention, operably linked to a nucleic acid insert encoding a polypeptide, and a polyadenylation signal sequence optionally operably linked thereto. It is contemplated that when the recombinant DNA is to be delivered to a recipient cell for expression therein, the sequence of the nucleic acid sequence may be modified so that the codons are optimized for the codon usage of the recipient species. For example, if the recombinant DNA is transfected into a recipient chicken cell, the sequence of the expressed nucleic acid insert is optimized for chicken codon usage. This may be determined from the codon usage of at least one, and preferably more than one, protein expressed in a chicken cell. For example, the codon usage may be determined from the nucleic acid sequences encoding the proteins ovalbumin, lysozyme, ovomucin and ovotransferrin of chicken.

In one embodiment of the recombinant DNA of the present invention, the nucleic acid insert encodes a therapeutic protein such as a human protein used as therapeutics wherein the sequence has been modified for codon optimization in an avian oviduct, e.g., a chicken oviduct. Optimization of the sequence for codon usage elevates the level of translation in avian eggs. As an example, the sequence (SEQ ID NO: 66) of the optimized human interferon alpha sequence is shown in FIG. 4.

In yet another embodiment of the present invention, the recombinant DNA comprises the isolated avian lysozyme gene expression control region operably linked to a nucleic acid encoding a therapeutic protein and a polyadenylation sequence, for example, the recombinant DNA having the nucleotide sequence of SEQ ID NO: 65, as shown in FIG. 3, or a variant thereof.

In still another embodiment of the present invention, the recombinant DNA comprises the isolated avian lysozyme gene expression control region operably linked to the nucleic acid encoding a polypeptide, and the chicken lysozyme 3′ domain SEQ ID NO: 69. In one embodiment of the present invention, the nucleic acid insert is SEQ ID NO: 66 encoding a human α2b interferon, and the recombinant DNA construct has the nucleic acid sequence of SEQ ID NO: 70.

The protein of the present invention may be produced in purified form by any known conventional techniques. For example, chicken cells may be homogenized and centrifuged. The supernatant is then subjected to sequential ammonium sulfate precipitation and heat treatment. The fraction containing the protein of the present invention is subjected to gel filtration in an appropriately sized dextran or polyacrylamide column to separate the proteins. If necessary, the protein fraction may be further purified by HPLC.

Recombinant Nucleic Acids, and Expression Thereof Under the Control of an Avian Lysozyme Promoter:

Another potentially useful application of the novel isolated lysozyme gene expression control region of the present invention is the possibility of increasing the amount of a heterologous protein present in a bird, (especially the chicken) by gene transfer. In most instances, a heterologous polypeptide-encoding nucleic acid insert transferred into the recipient animal host will be operably linked with the lysozyme gene expression control region, to allow the cell to initiate and continue production of the protein product. A recombinant DNA molecule of the present invention can be transferred into the extra-chromosomal or genomic DNA of the host.

The recombinant DNA nucleic acid molecules of the present invention can be delivered to cells using conventional recombinant DNA technology. The recombinant DNA molecule may be inserted into a cell to which the recombinant DNA molecule is heterologous (i.e. not normally present). Alternatively, as described more fully below, the recombinant DNA molecule may be introduced into cells which normally contain the recombinant DNA molecule, for example, to correct a deficiency in the expression of a polypeptide, or where over-expression of the polypeptide is desired.

For expression in heterologous systems, the heterologous DNA molecule is inserted into the expression system or vector of the present invention in proper sense orientation and correct reading frame. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences, including the novel isolated lysozyme gene expression control region.

U.S. Pat. No. 4,237,224 to Cohen and Boyer, hereby incorporated by reference in its entirety, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced to a cell by means of transformation and replicated in cultures, including eukaryotic cells grown in tissue culture.

One aspect of the present invention, therefore, is an expression vector suitable for delivery to a recipient cell for expression of the vector therein. It is contemplated to be within the scope of the present invention for the expression vector to comprise an isolated avian lysozyme gene expression control region operably linked to a nucleic acid insert encoding a polypeptide, and optionally a polyadenylation signal sequence. The expression vector of the present invention may further comprise a bacterial plasmid sequence, a viral nucleic acid sequence, or fragments or variants thereof that may allow for replication of the vector in a suitable host.

The novel isolated avian lysozyme gene expression control region of the present invention such as that included in SEQ ID NO: 67, and a polypeptide-encoding nucleic acid sequence operably linked thereto, such as, for example, SEQ ID NO: 66 or a derivative or truncated variant thereof, and optionally a polyadenylation signal sequence such as, for example, SEQ ID NO: 68 or the chicken lysozyme 3′ domain may be introduced into viruses such as vaccinia virus. Methods for making a viral recombinant vector useful for expressing a protein under the control of the lysozyme promoter are analogous to the methods disclosed in U.S. Pat. Nos. 4,603,112; 4,769,330; 5,174,993; 5,505,941; 5,338,683; 5,494,807; 4,722,848; Paoletti E., 1996, Proc. Natl. Acad. Sci., 93: 11349-11353; Moss, B., 1996, Proc. Natl. Acad. Sci. 93: 11341-11348; Roizman 1996, Proc. Natl. Acad. Sci. 93: 11307-11302; Frolov et al., 1996, Proc. Natl. Acad. Sci. 93: 11371-11377; Grunhaus et al., 1993, Seminars in Virology 3: 237-252 and U.S. Pat. Nos. 5,591,639; 5,589,466; and 5,580,859 relating to DNA expression vectors, inter alia; the contents of which are incorporated herein by reference in their entireties.

Recombinant viruses can also be generated by transfection of plasmids into cells infected with virus. Suitable vectors include, but are not limited to, viral vectors such as lambda vector system λgt11, λgt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC11, SV 40, pBluescript II SK+/− or KS+/− (see “Stratagene Cloning Systems” Catalog (1993) from Stratagene, La Jolla, Calif., which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see Studier, F. W. et. al., 1990, Use of T7 RNA Polymerase to Direct Expression of Cloned Genes in “Gene Expression Technology,” vol. 185, which is hereby incorporated by reference in its entirety) and any derivatives thereof. Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Maniatis et al., 1982, Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y., which is hereby incorporated by reference in its entirety.

A variety of host-vector systems may be utilized to express the protein-encoding sequence(s). Primarily, the vector system must be compatible with the host cell or host animal, such as an avian, that is used.

The use of eukaryotic recipient host cells permits partial or complete post-translational modification such as, but not only, glycosylation and/or the formation of the relevant inter- or intra-chain disulfide bonds. Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; vertebrate cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus) or avian embryonic cells inoculated with the recombinant nucleic acid. The expression elements of these vectors vary in their strength and specificities. Depending upon the host-vector system utilized, any one of a number of suitable transcription and translation elements can be used.

Once the novel isolated lysozyme gene expression control regions of the present invention have been cloned into a vector system, it is ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, and the like. Alternatively, it is contemplated that the incorporation of the DNA of the present invention into a recipient cell may be by any suitable method such as, but not limited to, viral transfer, electroporation, gene gun insertion, sperm mediated transfer to an ovum, microinjection and the like.

Another aspect of the present invention, therefore, is a method of expressing a heterologous polypeptide in a eukaryotic cell by transfecting the cell with a recombinant DNA comprising an avian lysozyme gene expression control region operably linked to a nucleic acid insert encoding a polypeptide and, optionally, a polyadenylation signal sequence, and culturing the transfected cell in a medium suitable for expression of the heterologous polypeptide under the control of the avian lysozyme gene expression control region.

In one embodiment of the method of the present invention, the recipient eukaryotic cell is derived from an avian. In one embodiment, the avian is a chicken.

Yet another aspect of the present invention is a eukaryotic cell transformed with an expression vector according to the present invention and described above. In one embodiment of the present invention, the transformed cell is a chicken oviduct cell and the nucleic acid insert comprises the chicken lysozyme gene expression control region, a nucleic acid insert encoding a human protein and codon optimized for expression in an avian cell, and a polyadenylation sequence.

It is contemplated that transfected cells according to the present invention may be transiently transfected, whereby the transfected recombinant DNA or expression vector may not be integrated into the genomic nucleic acid. It is further contemplated that the transfected recombinant DNA or expression vector may be stably integrated into the genomic DNA of the recipient cell, thereby replicating with the cell so that each daughter cell receives a copy of the transfected nucleic acid. It is still further contemplated for the scope of the present invention to include a transgenic animal producing a heterologous protein expressed from a transfected nucleic acid according to the present invention.

In one embodiment of the present invention, the transgenic animal is an avian selected from a turkey, duck, goose, quail, pheasant, ratite, an ornamental bird or a feral bird. In another embodiment, the avian is a chicken and the heterologous protein produced under the transcriptional control of the isolated avian lysozyme gene expression control region according to the present invention is produced in the white of an egg.

One particular aspect of the invention is directed to the use of retroviral constructs engineered to reduce or eliminate promoter interference. Promoter interference can be an undesired result that occurs when the function of a promoter interferes with the function of another promoter. In one embodiment, retroviral vectors designed to reduce or eliminate promoter interference can function by inactivation of the viral (e.g., LTR) promoter of a retroviral vector thereby functioning to reduce or eliminate LTR promoter interference of a promoter present in the vector which is operably linked to a coding sequence of interest. Such vectors are often referred to as self inactivating (i.e., SIN) vectors.

In another aspect, retroviral vectors can be engineered to reduce or eliminate promoter interference by removing from the vector, or not including in the vector in its initial construction, a selectable expression cassette, for example, which could be used for titering of the vector. Such vectors are referred to herein as SC negative vectors. SC negative vectors can still be titered, as is understood by a practitioner of skill in the art. However, such titering processes, which can involve determining relative cellular transfection frequency of the retroviral particles compared to that of standards of a known titer, are somewhat more difficult than titering using a selectable expression cassette. However, in some instances titering is not required for use of the retroviral vector to make a transgenic avian. In any case the lack of promoters of antibiotic resistance genes can eliminate the potential for promoter interference which can result from such promoters.

Self inactivating vectors and SC negative vectors are contemplated for use to reduce or eliminate promoter interference of any useful promoter which can be employed in transgenic avians such as chickens which produce exogenous proteins in the oviduct. For example, promoters which can preferentially express their gene product in oviduct cells or oviduct tissue are contemplated for use with SIN vectors and SC negative vectors. The invention contemplates the use of tissue specific promoters, constitutive promoters and inducible promoter for use with SIN vectors and SC negative vectors as disclosed herein. Examples of promoters which can be used with SIN vectors and SC negative vectors in accordance with the invention include but are not limited to, a cytomegalovirus (CMV) promoter, a rous-sarcoma virus (RSV) promoter, a β-actin promoter (e.g., a chicken β-actin promoter) a murine leukemia virus (MLV) promoter, a mouse mammary tumor virus (MMTV) promoter, an ovalbumin promoter, a lysozyme promoter, a conalbumin promoter, an ovomucoid promoter, an ovomucin promoter, and an ovotransferrin promoter. Optionally, the promoter may be a segment of at least one promoter region, such as a segment of the ovalbumin-, lysozyme-, conalbumin-, ovomucoid-, ovomucin-, and/or ovotransferrin promoter region. In one embodiment, the promoter is a combination or a fusion of one or more promoters or a fusion of a portion of one or more promoters such as ovalbumin-, lysozyme-, conalbumin-, ovomucoid-, ovomucin-, and ovotransferrin promoters with another promoter such as a viral promoter (e.g., an LTR promoter).

In one useful embodiment of the invention, a SIN vector is employed in a vector that is also an SC negative vector to produce a SIN/SC negative vector. The combination of SC negative vector and SIN vector can result in a vector with a substantially reduced amount of promoter interference compared to a vector that is only a SIN vector or only a negative vector.

In one useful embodiment, a SIN vector is produced in which a promoter that can inhibit transcription of a coding sequence operably linked to a lysozyme promoter of the invention (e.g., an LTR promoter) is inactivated, for example, by a deletion, insertion or transposition of all or part of the promoter sequence. In the case of the SIN/SC negative vector pALV-SIN-4.2-Lys-IFNa-2B, shown in FIG. 13, the 3′ LTR has a deletion in the enhancer such that when the retroviral region integrates, the 5′ LTR is inactivated. In addition, pALV-SIN-4.2-Lys-IFNa-2B also lacks an antibiotic resistance marker making it both a SC negative vector and a SIN vector. SIN vectors, SC vectors and SIN/SC negative vectors such as pALV-SIN-4.2-Lys-IFNa-2B are contemplated for application in any useful avian such as chicken, quail and turkey to produce chimeras including germ-line chimeras and progeny birds.

Viral Vector Cell Transformation:

An exemplary approach for the in vivo introduction of a nucleic acid encoding the subject novel isolated lysozyme gene expression control region into a cell is by use of a viral vector containing nucleic acid, e.g. a cDNA, encoding the gene product. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells that have taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. In one embodiment, recombinant retrovirus vectors can be constructed in a part of the retroviral coding sequence (gag, pol, env) that has been replaced by nucleic acid encoding a lysozyme gene expression control region, thereby rendering the retrovirus replication defective. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Ausubel et al, 1989, “Current Protocols in Molecular Biology,” Sections 9.10-9.14, Greene Publishing Associates, and other standard laboratory manuals. Examples of suitable retroviruses include, pLJ, pZIP, pWE and pEM, all of which are well known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include psiCrip, psiCre, psi2 and psiAm.

Avian retroviruses particularly useful in accordance with the present invention include, without limitation, Avian Leukemia/Leukosis Viruses (ALV), for example, and without limitation, RAV-0, RAV-1, RAV-2; Avian Sarcoma Viruses (ASV); Avian Sarcoma/Acute Leukemia Viruses (ASLV) including, without limitation, Rous Sarcoma Virus (RSV); Fujinami Sarcoma Viruses (FSV); Avian Myeloblastosis Viruses (AMV); Avian Erythroblastosis Viruses (AEV); Avian Myelocytomatosis Viruses (MCV), for example, and without limitation, MC29; Reticuloendotheliosis Viruses (REV), for example, and without limitation, Spleen Necrosis Virus (SNV). The invention also contemplates that the nucleotide sequence encoding a replication deficient retroviral vector can encode any useful retroviral vector, including, without limitation, retroviral vectors based upon Murine Leukemia Viruses (MLV); Molony Murine Sarcoma Viruses (MMSV); Moloney Murine Leukemia Viruses (MMLV); and lentiviruses (e.g., human immunodeficiency virus (HIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) and simian immunodeficiency virus (SIV). In one particularly useful embodiment, the retroviral vector employed herein is vector based on one or more of these or other retroviruses.

Furthermore, it is possible to limit the infection spectrum of retroviruses and consequently of retroviral-based vectors, by modifying the viral packaging proteins on the surface of the viral particle (see, for example PCT publications WO93/25234, WO94/06920, and WO94/11524). For instance, strategies for the modification of the infection spectrum of retroviral vectors include coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al., 1989, Proc. Natl. Acad. Sci. 86: 9079-9083; Julan et al., 1992, J. Gen. Virol. 73: 3251-3255 and Goud et al., 1983, Virology 163: 251-254) or coupling cell surface ligands to the viral env proteins (Neda et al., 1991, J. Biol. Chem. 266: 14143-14146) (all of which are incorporated herein by reference in their entireties). Coupling can be in the form of the chemical cross-linking with a protein or other moiety (e.g., lactose to convert the env protein to an asialoglycoprotein), as well as by generating fusion proteins (e.g., single-chain antibody/env fusion proteins). This technique, while useful to limit or otherwise direct the infection to certain tissue types, can also be used to convert an ecotropic vector into an amphotropic vector.

Another viral gene delivery system useful in the present invention utilizes adenovirus-derived vectors. The genome of an adenovirus can be manipulated such that it encodes a gene product of interest, but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle (see, for example, Berkner et al., 1988, BioTechniques 6: 616; Rosenfeld et al., 1991, Science 252: 43 1434; and Rosenfeld et al., 1992, Cell 68: 143-155, all of which are incorporated herein by reference in their entireties). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art. The virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Most replication-defective adenoviral vectors currently in use and therefore favored by the present invention are deleted for all or parts of the viral E1 and E3 genes but retain as much as 80% of the adenoviral genetic material (see, e.g., Jones et al., 1979, Cell 16:683; Berkner et al., supra; and Graham et al., 1991, pp. 109-127 in “Methods in Molecular Biology,” vol. 7, E. J. Murray, ed., Humana, Clifton, N.J., all of which are incorporated herein by reference in their entireties). Expression of an inserted DNA segment such as, DNA encoding a therapeutic protein can be under control of the exogenously added lysozyme gene expression control region sequences.

Yet another viral vector system useful for delivery of, for example, the subject avian lysozyme gene expression control region operably linked to a nucleic acid encoding a polypeptide, is the adeno-associated virus (AAV). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector, such as that described in Tratschin et al., 1985, Mol. Cell. Biol. 5: 3251-3260, can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see, for example, Hermonat et al., 1984, Proc. Natl. Acad. Sci. 81: 6466-6470; Tratschin et al., 1985, Mol. Cell. Biol. 4: 2072-2081; Wondisford et al., 1988, Mol. Endocrinol. 2: 32-39; Tratschin et al., 1984, J. Virol. 51: 611-619; and Flotte et al., 1993, J. Biol. Chem. 268: 3781-3790, all of which are incorporated herein by reference in their entireties).

Non-Viral Expression Vectors:

Most non-viral methods of gene transfer can rely on normal mechanisms used by eukaryotic cells for the uptake and intracellular transport of macromolecules. In preferred embodiments, non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the subject lysozyme gene expression control region and operably linked polypeptide-encoding nucleic acid by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes.

The invention provides for the use of non-viral methods of producing transgenic animals, such as transgenic avians, that contain nucleic acids of the invention (e.g., nucleic acids comprising a fragment of the lysozyme promoter operably linked to a nucleotide coding sequence). For example, the invention includes artificial chromosomes and integrase technologies for the production of transgenic avians. See, for example, U.S. patent application Ser. No. 11/362,064, filed Feb. 24, 2006, the disclosure of which is incorporated in its entirety herein by reference, and U.S. patent application Ser. No. 10/940,315, filed Sep. 14, 2004, the disclosure of which is incorporated in its entirety herein by reference.

In a representative embodiment, a nucleic acid comprising the novel isolated lysozyme gene expression control region of the present invention can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue for cellular delivery (Mizuno et al., 1992, NO Shinkei Geka 20: 547-551; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075, all of which are incorporated herein by reference in their entireties).

In similar fashion, the gene delivery system can comprise an antibody or cell surface ligand that is cross-linked with a gene binding agent such as polylysine (see, for example, PCT publications WO93/04701, WO92/22635, WO92/20316, WO92/19749, and WO92/06180, all of which are incorporated herein by reference in their entireties). It will also be appreciated that effective delivery of the subject nucleic acid constructs via receptor-mediated endocytosis can be improved using agents which enhance escape of gene from the endosomal structures. For instance, whole adenovirus or fusogenic peptides of the influenza HA gene product can be used as part of the delivery system to induce efficient disruption of DNA-containing endosomes (Mulligan et al., 1993, Science 260-926; Wagner et al., 1992, Proc. Natl. Acad. Sci. 89: 7934; and Christiano et al., 1993, Proc. Natl. Acad. Sci. 90: 2122, all of which are incorporated herein by reference in their entireties). It is further contemplated that a recombinant DNA molecule comprising the novel isolated lysozyme gene expression control region of the present invention may be delivered to a recipient host cell by other non-viral methods including by gene gun, microinjection, sperm-mediated transfer, or the like.

Trans Genic Animals:

Another aspect of the present invention concerns transgenic animals, in particular avians such as chickens, having a transgene comprising the novel isolated lysozyme gene expression control region of the present invention and which preferably (though optionally) express a heterologous gene in one or more cells in the animal. Suitable methods for the generation of transgenic avians having heterologous DNA incorporated therein are described, for example, in WO 99/19472 to Ivarie et al.; WO 00/11151 to Ivarie et al.; and WO 00/56932 to Harvey et al., all of which are incorporated herein by reference in their entirety. Particularly useful methods of making transgenic avians, such as chickens which employ fragments of the avian lysozyme promoter of the invention are disclosed in U.S. patent application Ser. No. 11/167,052, filed Jun. 24, 2005, the disclosure of which is incorporated in its entirety herein by reference.

In certain embodiments of the present invention, the expression of the transgene may be restricted to specific subsets of cells, tissues or developmental stages utilizing, for example, cis-acting sequences acting on the lysozyme gene expression control region of the present invention and which control gene expression in the desired pattern. Tissue-specific regulatory sequences and conditional regulatory sequences can be used to control expression of the transgene in certain spatial patterns. Moreover, temporal patterns of expression can be provided by, for example, conditional recombination systems or prokaryotic transcriptional regulatory sequences. The inclusion of a 5′ MAR region in the novel isolated lysozyme gene expression control region of the present invention may allow the heterologous expression unit to escape the chromosomal positional effect (CPE) and therefore be expressed at a more uniform level in transgenic tissues that received the transgene by a route other than through germ line cells.

One embodiment of the present invention, therefore, is a transgenic avian having a heterologous polynucleotide sequence comprising a nucleic acid insert encoding the heterologous polypeptide and operably linked to the novel isolated avian lysozyme gene expression control region, the lysozyme gene expression control region comprising at least one 5′ matrix attachment region, an intrinsically curved DNA region, at least one transcription enhancer, a negative regulatory element, at least one hormone responsive element, at least one avian CR1 repeat element, and a proximal lysozyme promoter and signal peptide-encoding region.

In an embodiment of the present invention, the transgenic avian is selected from a chicken, a turkey, a duck, a goose, a quail, a pheasant, a ratite, an ornamental bird or a feral bird.

In another embodiment of the present invention, the transgenic avian is a chicken.

In still another embodiment of the transgenic avian of the present invention, the transgenic avian includes an avian lysozyme gene expression control region comprising the nucleic acid sequence in SEQ ID NO: 67, or a variant thereof.

In yet another embodiment of the transgenic avian of the present invention, the transgenic avian further comprises a polyadenylation signal sequence.

In still yet another embodiment of the transgenic avian of the present invention, the polyadenylation signal sequence is derived from the SV40 virus.

In an embodiment of the transgenic avian of the present invention, the polyadenylation signal sequence comprises the nucleic acid sequence in SEQ ID NO: 68, or variant thereof.

In still another embodiment of transgenic avian of the present invention, the transgenic avian comprises the chicken lysozyme 3′ domain having the nucleic acid sequence of SEQ ID NO: 69.

In another embodiment of the transgenic avian of the present invention, the nucleic acid insert encoding a polypeptide has a codon complement optimized for protein expression in an avian.

In yet another embodiment of the transgenic avian of the present invention, the nucleic acid insert encodes an interferon α.

In still another embodiment of the transgenic avian of the present invention, the nucleic acid insert encoding an interferon α2b polypeptide comprises the sequence in SEQ ID NO: 66, or a variant thereof.

In one embodiment of the transgenic avian of the present invention, the transgenic avian comprises the nucleotide sequence in SEQ ID NO: 65, or a variant thereof.

In yet another embodiment of the transgenic avian of the present invention, the transgenic avian comprises the nucleotide sequence in SEQ ID NO: 70, or a variant thereof. In another embodiment of the transgenic avian of the present invention, the transgenic avian produces the heterologous polypeptide in the serum or an egg white.

In another embodiment of the transgenic avian of the present invention, the transgenic avian produces the heterologous polypeptide in an egg white.

Therapeutic Proteins

The invention can be used to produce a wide range of desired therapeutic proteins such as fusion proteins, growth hormones, cytokines, structural proteins and enzymes including human growth hormone, interferon, lysozyme, and β-casein. Other possible proteins contemplated for production as disclosed herein include, but are not limited to, albumin, α-1 antitrypsin, antithrombin III, collagen, factors VIII, IX, X (and the like), fibrinogen, hyaluronic acid, insulin, lactoferrin, protein C, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), tissue-type plasminogen activator (tPA), somatotropin, and chymotrypsin. Modified immunoglobulins and antibodies, including immunotoxins which bind to surface antigens on human tumor cells and destroy them, can also be produced as disclosed herein.

Other specific examples of therapeutic proteins which may be produced as disclosed herein include, without limitation, factor VIII, b-domain deleted factor VIII, factor VIIa, factor IX, anticoagulants; hirudin, alteplase, tpa, reteplase, tpa, tpa—3 of 5 domains deleted, insulin, insulin lispro, insulin aspart, insulin glargine, long-acting insulin analogs, hgh, glucagons, tsh, follitropin-beta, fsh, gm-csf, pdgh, ifn alpha2, ifn alpha2a, ifn alpha2b, inf-apha, inf-beta 1b, ifn-beta 1a, ifn-gamma1b, il-2, il-11, hbsag, ospa, murine mab directed against t-lymphocyte antigen, murine mab directed against tag-72, tumor-associated glycoprotein, fab fragments derived from chimeric mab directed against platelet surface receptor gpII(b)/III(a), murine mab fragment directed against tumor-associated antigen ca125, murine mab fragment directed against human carcinoembryonic antigen, cea, murine mab fragment directed against human cardiac myosin, murine mab fragment directed against tumor surface antigen psma, murine mab fragments (fab/fab2 mix) directed against hmw-maa, murine mab fragment (fab) directed against carcinoma-associated antigen, mab fragments (fab) directed against nca 90, a surface granulocyte nonspecific cross reacting antigen, chimeric mab directed against cd20 antigen found on surface of b lymphocytes, humanized mab directed against the alpha chain of the il2 receptor, chimeric mab directed against the alpha chain of the il2 receptor, chimeric mab directed against tnf-alpha, humanized mab directed against an epitope on the surface of respiratory synctial virus, humanized mab directed against her 2, human epidermal growth factor receptor 2, human mab directed against cytokeratin tumor-associated antigen anti-ctla4, chimeric mab directed against cd 20 surface antigen of b lymphocytes dornase-alpha dnase, beta glucocerebrosidase, tnf-alpha, il-2-diptheria toxin fusion protein, tnfr-lgg fragment fusion protein laronidase, dnaases, alefacept, darbepoetin alfa (colony stimulating factor), tositumomab, murine mab, alemtuzumab, rasburicase, agalsidase beta, teriparatide, parathyroid hormone derivatives, adalimumab (Igg1), anakinra, biological modifier, nesiritide, human b-type natriuretic peptide (hbnp), colony stimulating factors, pegvisomant, human growth hormone receptor antagonist, recombinant activated protein c, omalizumab, immunoglobulin e (lge) blocker, lbritumomab tiuxetan, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigmentary hormones, somatomedin, erythropoietin, luteinizing hormone, chorionic gonadotropin, hypothalmic releasing factors, antidiuretic hormones, prolactin and thyroid stimulating hormone.

The invention, includes methods for producing multimeric proteins including immunoglobulins, such as antibodies, and antigen binding fragments thereof. Thus, in one embodiment of the present invention, the multimeric protein is an immunoglobulin, wherein the first and second heterologous polypeptides are immunoglobulin heavy and light chains respectively.

In certain embodiments, an immunoglobulin polypeptide encoded by the transcriptional unit of at least one expression vector may be an immunoglobulin heavy chain polypeptide comprising a variable region or a variant thereof, and may further comprise a D region, a J region, a C region, or a combination thereof. An immunoglobulin polypeptide encoded by an expression vector may also be an immunoglobulin light chain polypeptide comprising a variable region or a variant thereof, and may further comprise a J region and a C region. The present invention also contemplates multiple immunoglobulin regions that are derived from the same animal species, or a mixture of species including, but not only, human, mouse, rat, rabbit and chicken. In certain embodiments, the antibodies are human or humanized.

In other embodiments, the immunoglobulin polypeptide encoded by at least one expression vector comprises an immunoglobulin heavy chain variable region, an immunoglobulin light chain variable region, and a linker peptide thereby forming a single-chain antibody capable of selectively binding an antigen.

Examples of therapeutic antibodies that may be produced in methods of the invention include, but are not limited, to HERCEPTIN™ (Trastuzumab) (Genentech, CA) which is a humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer; REOPRO™ (abciximab) (Centocor) which is an anti-glycoprotein IIb/IIIa receptor on the platelets for the prevention of clot formation; ZENAPAX™ (daclizumab) (Roche Pharmaceuticals, Switzerland) which is an immunosuppressive, humanized anti-CD25 monoclonal antibody for the prevention of acute renal allograft rejection; PANOREX™ which is a murine anti-17-IA cell surface antigen IgG2a antibody (Glaxo Wellcome/Centocor); BEC2 which is a murine anti-idiotype (GD3 epitope) IgG antibody (ImClone System); IMC-C225 which is a chimeric anti-EGFR IgG antibody (ImClone System); VITAXIN™ which is a humanized anti-αVβ3 integrin antibody (Applied Molecular Evolution/MedImmune); Campath 1H/LDP-03 which is a humanized anti CD52 IgG1 antibody (Leukosite); Smart M195 which is a humanized anti-CD33 IgG antibody (Protein Design Lab/Kanebo); RITUXAN™ which is a chimeric anti-CD2O IgG1 antibody (IDEC Pharm/Genentech, Roche/Zettyaku); LYMPHOCIDE™ which is a humanized anti-CD22 IgG antibody (Immunomedics); ICM3 is a humanized anti-ICAM3 antibody (ICOS Pharm); IDEC-114 is a primate anti-CD80 antibody (IDEC Pharm/Mitsubishi); ZEVALIN™ is a radiolabelled murine anti-CD20 antibody (IDEC/Schering AG); IDEC-131 is a humanized anti-CD40L antibody (IDEC/Eisai); IDEC-151 is a primatized anti-CD4 antibody (IDEC); IDEC-152 is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is a humanized anti-complement factor 5 (CS) antibody (Alexion Pharm); D2E7 is a humanized anti-TNF-α antibody (CATIBASF); CDP870 is a humanized anti-TNF-α Fab fragment (Celltech); IDEC-151 is a primatized anti-CD4 IgG1 antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4 is a human anti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 is a humanized anti-TNF-α IgG4 antibody (Celltech); LDP-02 is a humanized anti-α4β7 antibody (LeukoSite/Genentech); OrthoClone OKT4A is a humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVA™ is a humanized anti-CD40L IgG antibody (Biogen); ANTEGREN™ is a humanized anti-VLA-4 IgG antibody (Elan); and CAT-152, a human anti-TGF-β₂ antibody (Cambridge Ab Tech).

The present invention is further illustrated by the following examples, which are provided by way of illustration and should not be construed as limiting. The contents of all references, published patents and patents cited throughout the present application are hereby incorporated by reference in their entireties.

Example 1 Construction of Lysozyme Promoter Plasmids

The chicken lysozyme gene expression control region was isolated by PCR amplification. Ligation and reamplification of the fragments thereby obtained yielded a contiguous nucleic acid construct comprising the chicken lysozyme gene expression control region operably linked to a nucleic acid sequence optimized for codon usage in the chicken (SEQ ID NO: 66) and encoding a human interferon α2b polypeptide optimized for expression in an avian cell.

White Leghorn Chicken (Gallus gallus) genomic DNA was PCR amplified using the primers 5pLMAR2 (SEQ ID NO: 1) (see FIG. 1) and LE-6.1 kbrev1 (SEQ ID NO: 2) in a first reaction, and Lys-6.1 (SEQ ID NO: 3) and LysE1rev (SEQ ID NO: 4) as primers in a second reaction. PCR cycling steps were: denaturation at 94° C. for 1 minute; annealing at 60° C. for 1 minute; extension at 72° C. for 6 minutes, for 30 cycles using TAQ PLUS PRECISION™ DNA polymerase (Stratagene, LaJolla, Calif.). The PCR products from these two reactions were gel purified, and then united in a third PCR reaction using only 5pLMAR2 (SEQ ID NO: 1) and LysE1rev (SEQ ID NO: 4) as primers and a 10-minute extension period. The resulting DNA product was phosphorylated, gel-purified, and cloned into the EcoR V restriction site of the vector pBluescript KS, resulting in the plasmid p12.0-lys.

p12.0-lys was used as a template in a PCR reaction with primers 5pLMAR2 (SEQ ID NO: 1) and LYSBSU (SEQ ID NO: 5) and a 10 minute extension time. The resulting DNA was phosphorylated, gel-purified, and cloned into the EcoR V restriction site of pBluescript KS, forming plasmid p12.0lys-B.

p12.0lys-B was restriction digested with Not I and Bsu36 I, gel-purified, and cloned into Not I and Bsu36 I digested pCMV-LysSPIFNMM, resulting in p12.0-lys-LSPIFNMM. p12.0-lys-LSPIFNMM was digested with Sal I and the SalI to NotI primer (SEQ ID NO: 6) was annealed to the digested plasmid, followed by Not I digestion. The resulting 12.5 kb Not I fragment, comprising the lysozyme promoter region linked to IFNMAGMAX-encoding region and an SV40 polyadenylation signal sequence, was gel-purified and ligated to Not I cleaved and dephosphorylated pBluescript KS, thereby forming the plasmid pAVIJCR-A 115.93.1.2. The lysozyme promoter/IFN construct contained in plasmid pAVIJCR-A 115.93.1.2 was sequenced as described in Example 3.

Example 2 Construction of Plasmids which Contain the 3′ Lysozyme Domain

The plasmid pAVIJCR-A 115.93.1.2 was restriction digested with FseI and blunt-ended with T4 DNA polymerase. The linearized, blunt-ended pAVIJCR-A 115.93.1.2 plasmid was then digested with XhoI restriction enzyme, followed by treatment with alkaline phosphatase. The resulting 15.4 kb DNA band containing the lysozyme 5′ matrix attachment region (MAR) and −12.0 kb lysozyme promoter driving expression of a human interferon was gel purified by electroelution.

The plasmid pIIIilys was restriction digested with MluI, then blunt-ended with the Klenow fragment of DNA polymerase. The linearized, blunt-ended pIIIilys plasmid was digested with XhoI restriction enzyme and the resulting 6 kb band containing the 3′ lysozyme domain from exon 3 to the 3′ end of the 3′ MAR was gel purified by electroelution. The 15.4 kb band from pAVIJCR-A115.93.1.2 and the 6 kb band from pIIIilys were ligated with T4 DNA ligase and transformed into STBL4 cells (Invitrogen Life Technologies, Carlsbad, Calif.) by electroporation. The resulting 21.3 kb plasmids from two different bacterial colonies were named pAVIJCR-A212.89.2.1 and pAVIJCR-A212.89.2.3 respectively.

Example 3 Sequencing Reactions

Plasmid DNA (pAVIJCR-A115.93.1.2) produced as described in Example 1 was purified with QIAGEN™ columns (Qiagen, Valencia, Calif.). Sequencing reactions were performed according to the Applied Biosystems (Foster City, Calif.) protocol for BIGDYE™ Terminators, version 2.0, using an ABI 373 Stretch sequencer. Sequencing primers used are listed in FIG. 1, and a schematic diagram illustrating the sequencing reactions using the different primers is shown in FIG. 2. Sequence data was analyzed with SEQUENCHER™ software, version 4.0 (Gene Codes Corp., Ann Arbor, Mich.).

Example 4 Complete Lysozyme Promoter and IFNMAGMAX Sequences

The complete nucleotide sequence (SEQ ID NO: 65), shown in FIG. 3, of the 12.5 kb chicken lysozyme promoter region/IFNMAGMAX construct spans the 5′ matrix attachment region (5′ MAR), through the lysozyme signal peptide, to the sequence encoding the gene IFNMAGMAX and the subsequent polyadenylation signal sequence. The IFNMAGMAX nucleic acid sequence (SEQ ID NO: 66), shown in FIG. 4, encoded human interferon α2b (IFN) that had been synthesized based on a codon usage table compiled from the four most abundantly expressed hen egg white proteins ovalbumen, ovotransferrin, ovomucoid and lysozyme. The expressed IFN α2b sequence within plasmid pAVIJCR-A1 15.93.1.2 functioned as a reporter gene for lysozyme promoter activity. This plasmid construct may also be used for production of interferon α2b in the egg white of transgenic chickens. The isolated sequence of the 11.94 kb chicken lysozyme promoter region (SEQ ID NO: 67) alone is shown in FIG. 5. The sequence of the SV40 polyadenylation signal sequence (SEQ ID NO: 68) is shown in FIG. 6.

Example 5 Basic Local Alignment Search Tool (BLAST) Analysis of the Complete Lysozyme Promoter Sequence (SEQ ID NO: 65)

The complete 12.5 kb lysozyme promoter/IFNMAGMAX sequence (SEQ ID NO: 65) was submitted to the National Center for Biotechnology Information for BLAST alignments with database sequences. Percent identities between the lysozyme promoter sequence (SEQ ID NO: 67, included within SEQ ID NO: 65) and corresponding known lysozyme promoter features are shown in Table II below:

TABLE II BLAST Results of the Complete 12.0 kb Lysozyme Promoter Sequence Coordinates in GenBank accession Description of DNA element this sequence number % identity 5′ matrix attachment region 1-237, 261-1564 AJ277960 96 5′ matrix attachment region 1-237, 261-1564 X98408 96 5′ matrix attachment region 1564-1912 X84223 99 1930-2015 Intrinsically curved DNA 2011-2671 X52989 98 Transcription enhancer (−6.1 kb) 5848-5934 Grewal et al., 1992 100 Transcription enhancer (E−2.7 kb) 9160-9329 X05461 98 Negative regulatory element 9325-9626 X05463 98 Hormone response element 9621-9666 X12509 99  9680-10060 CR1 chicken repeat element  10576-10821, U88211, K02907 87 10926-11193 Transcription enhancer (E−0.2 kb) 11655-11797 X05462 100 Proximal promoter and 11563-11877 M12532 100 lysozyme signal peptide Proximal promoter and 11424-11938 J00886 99 lysozyme signal peptide

Features that have been previously identified as individual elements isolated from other component elements of the lysozyme promoter region include the 5′ MAR, three transcription enhancers, a hormone-responsive element, and a chicken repeat 1 (CR1) element. The IFNMAGMAX sequence (SEQ ID NO: 66) extended from nucleotide positions 11946 to 12443 of SEQ ID NO: 65, shown in FIG. 3.

Example 6 Expression in Transfected Cultured Avian Oviduct Cells of Human Interferon α2b Regulated by the 12 kb Lysozyme Promoter

The oviduct was removed from a Japanese quail (Coturnix coturnix japonica) and the magnum portion minced and enzymatically dissociated with 0.8 mg/ml collagenase (Sigma Chemical Co., St. Louis, Mo.) and 1.0 mg/ml dispase (Roche Molecular Biochemicals, Indianapolis, Ind.) by shaking and titurating for 30 minutes at 37° C. The cell suspension was then filtered through sterile surgical gauze, washed three times with F-12 medium (Life Technologies, Grand Island, N.Y.) by centrifugation at 200×g, and resuspended in OPTIMEM™ (Life Technologies) such that the OD₆₀₀ was approximately 2. Cell suspension (300 μl) was plated per well of a 24-well dish. For each transfection, 2.5 μl of DMRIE-C liposomes (Life Technologies) and 1 μg of DNA were preincubated for 15 minutes at room temperature in 100 μl of OPTIMEM™, and then added to the oviduct cells. Cells with DNA/liposomes were incubated for 5 hours at 37° C. in 5% CO₂. Next, 0.75 ml of DMEM (Life Technologies) supplemented with 15% fetal bovine serum (FBS) (Atlanta Biologicals, Atlanta, Ga.), 2× penicillin/streptomycin (Life Technologies), 10⁻⁶ M insulin (Sigma), 10⁻⁸ M β-estradiol (Sigma), and 10⁻⁷ M corticosterone (Sigma) was added to each well, and incubation was continued for 72 hours. Medium was then harvested and centrifuged at 110×g for 5 minutes. The supernatant was analyzed by ELISA for human interferon α2b content.

The human interferon α2b contents of medium derived from cultured oviduct cells transfected with either the −12.0 kb IFN plasmid (pAVIJCR-A115.93.1.2) or the negative control plasmid pCMV-EGFP as shown in FIG. 7. Bars to the right of the figure represent the standards for the IFN ELISA.

Example 7 Transfection of Chicken HD11 Cells with pAVIJCR-A212.89.2.1 and pAVIJCR-A212.89.2.3

Chicken cells transfected with plasmids having the 3′ lysozyme domain linked to a nucleic acid expressing human α2b interferon express the heterologous polypeptide. Chicken myelomonocytic HD11 cells were transfected with plasmid pAVIJCR-A212.89.2.1 and pAVIJCR-A212.89.2.3 to test the functionality of the plasmids. One million HD11 cells were plated per each well of a 24-well dish. The next day, HD11 cells were transfected with 1 μg of plasmid DNA per 4 μl of LipofectAMINE 2000 (Invitrogen Life Technologies). For comparison, independent wells were also transfected with the parent vector pAVIJCR-A 15.93.1.2. After 5 hours of transfection, the cell medium was changed with fresh medium. 48 hours later, cell medium was harvested by centrifugation at 110×g for 5 min and assayed for human interferon by ELISA (PBL Biomedicals, Flanders, N.J.).

The transfected cells expressed the heterologous human α2b interferon at least to the level seen with a plasmid not having the 3′ lysozyme domain operably linked to the human α2b interferon encoding nucleic acid, as shown in FIG. 10.

Example 8 Expression of Human α2b Interferon in a Transgenic Avian Platform

The plasmid pAVIJCR-A115.93.1.2 (containing the −12.0 kb lysozyme promoter controlling expression of human interferon α-2b) was purified with a Qiagen Plasmid Maxi Kit (Qiagen, Valencia, Calif.), and 100 micrograms of the plasmid were restriction digested with NotI restriction enzyme. The digested DNA was phenol/CHCl₃ extracted and ethanol precipitated. Recovered DNA was resuspended in 1 mM Tris-HCl (pH 8.0) and 0.1 mM EDTA, then placed overnight at 4° C. DNA was quantified by spectrophotometry and diluted to the appropriate concentration. DNA samples were resuspended in 0.25 M KCl and bound with a SV40 T antigen nuclear localization signal peptide (NLS peptide, amino acid sequence CGGPKKKRKVG-NH₂; SEQ ID NO: 71) by adding the NLS at a peptide:DNA molar ratio of 100:1 (Collas and Alestrom, 1996, Mol. Reprod. Develop. 45: 431-438; the contents of which is incorporated by reference in its entirety).

Cytoplasmic Microinjection of DNA. Approximately two nanoliters of DNA were injected into the germinal disk of stage I White Leghorn embryos obtained two hours after oviposition of the previous egg. DNA amounts per injection ranged from 10 picograms to 400 picograms. Injected embryos were surgically transferred to recipient hens via ovum transfer according to the method of Christmann et al (PCT/US01/26723 to Christmann et al; the contents of which is hereby incorporated by reference in its entirety) Analysis of chick blood DNA by PCR for IFN transgene: Whole blood from one week old chicks was collected with heparinized capillary tubes. Red blood cell (RBC) nuclei were released and washed with lysis buffer solution. DNA's from RBC nuclei were extracted by digestion with proteinase K (1 mg/ml) and precipitated with ethanol. Purified DNA was resuspended in 1 mM Tris-HCl (pH 8.0) and 0.1 mM EDTA and quantitated. Genomic DNA samples were analyzed by PCR using primers LYS051 (SEQ ID NO.: 72) for 5′-TGCATCCTTCAGCACTTGAG-3′ and IFN-3 (SEQ ID NO: 73) for 5′-AACTCCTCTTGAGGAAAGCC-3′. This primer set amplifies a 584 bp region of the transgene carried by the pAVIJCR-A 115.93.1.2 plasmid. Three hundred nanograms of genomic DNA were added to a 50 μl reaction mixture (1× Promega PCR Buffer with 1.5 mM MgCl₂, 200 μM of each dNTP, 5 μM primers) and 1.25 units of Taq DNA polymerase (Promega). The reaction mixtures were heated for 4 minutes at 94° C. and then amplified for 34 cycles at 94° C. for 1 min, 60° C. for 1 min and 72° C. for 1 min. The samples were heated in a final cycle for 4 minutes at 72° C. PCR products were detected on a 0.8% agarose gel with ethidium bromide staining. Human interferon α-2b expression in chick blood by ELISA: One week after hatch, blood was collected from chicks using heparinized capillary tubes; added to an equal volume of phosphate buffered saline and centrifuged at 200×g. 100 microliters of the supernatant were assayed by human IFN ELISA (PBL Biomedical Laboratories, New Brunswick, N.J.). Human interferon α-2b expression in egg white of transgenic hens: once hens reached sexual maturity and began to lay (approximately 22-24 weeks of age), eggs were collected and egg white assayed by ELISA using human IFN ELISA (PBL Biomedical Laboratories, New Brunswick, N.J.) according to manufacturer's instructions. Results of PCR and ELISA analysis of blood and egg white: Table III below summarizes results of PCR and ELISA analysis.

TABLE III Analysis of Transgene presence and Interferon Expression PCR ELISA ELISA Bird # Method (Blood) (Blood) (egg white) # Birds Tested 8305 −NLS + + NA (male) 8340 −NLS − − + 69 (2.5%) AA123 +NLS + + NA (immature) AA61 +NLS + + ″ AA105 +NLS − + ″ AA115 +NLS + − ″ 43 (9%)   −NLS: DNA injected without NLS peptide; +NLS: DNA injected with NLS peptide; NA: not applicable

As shown in Table III, one bird (#8305) of 69 produced using microinjection of DNA without the NLS peptide, was positive for both presence of the transgene and expression of interferon in the blood. Because this bird is a male, he will be bred to a non-transgenic hen to examine germline transmission of the transgene. FIG. 11 demonstrates expression of human interferon in the blood of #8305, as compared to standards. FIG. 12 illustrates PCR results from the serum for several birds, including #8305, obtained at different intervals after hatch. As can be seen in lanes 4, 5, 11, and 12, positive signal was seen indicating the presence of the transgene at two different collection periods.

Other positives were seen in birds produced under microinjection of DNA covalently linked to the NLS peptide as described above. Table III illustrates 4 birds (AA123, AA61, AA105 and AA115) out of 43 tested that were PCR positive, ELISA positive or both. Expression levels of human IFN in AA61, as compared to standards, is also illustrated in FIG. 11. Males will be bred to determine germline transmission, and eggs collected from transgenic females to assay for IFN expression, as described above, as chicks reach sexual maturity.

Example 9 Expression of a Human Monoclonal Antibody (Mab) in a Transgenic Avian Platform

Transgenic chickens were produced as described in Example 8 above, except that two distinct constructs were coinjected into Stage 1 embryos. The constructs comprised the 12 kb lysozyme promoter, as described above in Example 4, driving either a heavy chain or light chain of a human monoclonal antibody against CTLA-4 (WO 01/14424 A2 to Korman et al.; the contents of which is incorporated herein in its entirety). ELISA analysis of serum, conducted as described above in Example 8, is summarized below:

TABLE IV ELISA analysis of Mab expression in hatched birds: ELISA ELISA # Birds Bird # Method (serum) (egg white) Tested 214 +NLS + NA (immature) 228 +NLS + ″ 13 +NLS: DNA injected with NLS peptide; NA: not available Results indicate that two birds of the thirteen tested to date, #214 and #228, are positive for Mab expression in the serum.

Example 10 Production of Transgenic Quail Containing pALV-SIN-4.2-Lys-IFNa-2B in their Genome

The vector pALV-SIN-4.2-Lys-IFNa-2B (shown in FIG. 13) was constructed and used to produce transgenic Quail. The sequence of pALV-SIN-4.2-Lys-IFNa-2B is shown in SEQ ID NO: 75. The 4.2 Kb lysozyme promoter spans from nucleotides 4810 to 9008 of SEQ ID NO: 75. The lysozyme signal peptide coding sequence spans from nucleotides 9037 to 9090 of SEQ ID NO: 75. The interferon alpha 2b coding sequence spans from nucleotides 9091 to 9585 of SEQ ID NO: 75. Other components of the sequence include LTRs spanning from nucleotides 4000 to 4345 and from nucleotides 725 to 897.

pALV-SIN-4.2-Lys-IFNa-2B can be constructed by a variety of methods which are apparent to a practitioner of skill in the art. However, the method believed to be the best for making the vector is as follows: A 3427 bp region of pNLB-CMV-IFN-alpha2B (disclosed in U.S. patent application Ser. No. 11/167,052, filed Jun. 24, 2005, the disclosure of which is incorporated in its entirety herein by reference) is PCR amplified using primers ATGCGCGCATTGGTAATTGATCGGCTGG (Primer ALV-SIN-1, SEQ ID NO: 76) and ATATGCGGCCGCGGTACCGCCCGGGCATCGATATCAAGCTTACGGTTCACTA AACGAGCTCTGCTTATATAGACCTCCCA (Primer ALV-SIN-2, SEQ ID NO: 77). The product is digested with BssHII and Not I resulting in a 3428 bp fragment which can be isolated by gel purification. A 1436 bp region of pNLB-CMV-IFN-alpha2B is PCR amplified with primers ATATGCGGCCGCGTCGACGGCCGGCCAGATCTGCTGAGCCGGTCGCTACCAT TACCAGT (Primer ALV-SIN-3, SEQ ID NO: 78) and ATACGCGTATTCCCTAACGATCACGTCG (Primer ALV-SIN-4, SEQ ID NO: 79). The resulting product is digested with Not I and Mlu I yielding a 1438 bp fragment which is isolated by gel purification. A Bluescript II SK vector containing a BssHII stuffer fragment is digested with BssHII resulting in a linearized Bluescript vector of 2788 bp which is gel purified and then ligated to the 3428 bp and 1438 bp PCR products to yield JCR.A108.49.5.24.

JCR.A108.49.5.24 is digested with Hind III and the resulting 6823 bp fragment is circularized by ligation to yield JCR.A108.76.1.1.

A 1175 bp region of JCR.A108.76.1.1 is PCR amplified with primers CTGAAGTGTAAGGAATGTAAG (Primer ALV-SIN-5, SEQ ID NO: 80) and GCGCGTCTCATCCCCCTCCCTATGCAAAAG (Primer ALV-SIN-6, SEQ ID NO: 81) and the resulting fragment is digested with Blp I and Esp3I producing a 1030 bp fragment which is isolated by gel purification. A 660 bp region of JCR.A108.76.1.1 is PCR amplified with primers GGGCGTCTCAGGGACGGATTGGACGAACCACTGAATT (Primer ALV-SIN-7, SEQ ID NO: 82) and TTAGTGCTTTACGGCACCTC (Primer ALV-SIN-8, SEQ ID NO: 83) and digested with Esp3I and DraIII resulting in a 596 bp fragment which is isolated by gel purification. JCR.A108.76.1.1 is digested with DraIII and Blp I and the 5024 bp linear vector is ligated to the 1030 and 596 bp PCR fragments to produce pALV-SIN.

pALV-SIN is digested with BamHI and the 4795 bp linear vector is isolated by gel purification. A 4815 bp region of JCR.115.93.1.2 is PCR amplified with primers GACGGATCCGATACCGTCCCTATTTTTGTGTTTGCTTC (Primer ALV-SIN-9, SEQ ID NO: 84) and TAACGGATCCTAGACTTTTTACTCCTTAGA (Primer ALV-SIN-10, SEQ ID NO: 85) and is digested with BamHI. The resulting 4802 fragment is ligated to the 4795 bp linear pALV-SIN to create pALV-SIN-4.0-Lys-IFNa-2B.

Transduction particles of the vector pALV-SIN-4.2-Lys-IFNa-2B were produced in fibroblast cells as disclosed in U.S. patent application Ser. No. 11/542,093, filed Oct. 3, 2006 titled: Rapid Production of High Titer Virus, the disclosure of which is incorporated in its entirety herein by reference.

Fertilized Japanese quail eggs were windowed essentially according to the Speksnijder procedure (U.S. Pat. No. 5,897,998, the disclosure of which is incorporated in its entirety herein by reference) 80 eggs were injected in the subgerminal cavity with about 7×10⁴ pALV-SIN-4.2-Lys-IFNa-2B transducing particles per egg of which 16 chicks hatched. Eggs hatched 18 days after injection and human IFN levels were measured by IFN ELISA from serum samples collected from chicks 12 weeks after hatch. None were positive for the IFN protein in the serum.

In order to identify G0 quail which contained the interferon alpha 2 coding sequence containing transgene in their genome, DNA was extracted from blood of the birds and the DNA samples were subjected to Taqman® analysis on a 7700 Sequence Detector (Perkin Elmer). Quail No. 4 was positive for the transgene.

Eggs from eight G0 quail were tested for the presence of the IFN protein in the egg white by ELISA. Quail No. 4 was found to have significant levels of IFN in egg white from her eggs. FIG. 14 shows a bar graph illustrating expression levels of IFN in the egg white of Quail No. 4. Quail No. 4 expressed IFN-alpha-2 at 0.45 micrograms per ml of egg white, which is a high level of expression for a G0 avian. There was no interferon alpha 2 detected in the blood of Quail No. 4. This is particularly significant since the lack of circulating recombinant protein can facilitate recombinant protein production. This is because, for example, in certain instances the recombinant (exogenous) protein may be harmful to the development or health of the avian when present in the blood.

Example 11 Production of Transgenic Avians Containing pALV-SIN-6.5-Lys-IFNa-2B

The 4.2 kb lysozyme promoter of vector pALV-SIN-4.2-Lys-IFNa-2B is removed and replaced with a 6.5 kb lysozyme promoter corresponding to nucleotides 7665 to 11863 of SEQ ID NO: 67 using standard methodologies known to practitioners of skill in the art, resulting in pALV-SIN-6.5-Lys-IFNa-2B. Transduction particles of the new vector pALV-SIN-6.5-Lys-IFNa-2B are produced as disclosed in U.S. patent application Ser. No. 11/542,093, filed Oct. 3, 2006.

Fertilized chicken eggs or Japanese quail eggs are windowed and about 7×10⁴ pALV-SIN-6.5-Lys-IFNa-2B transducing particles are injected into the subgerminal cavity of each egg. Eggs hatch 21 or 18 days after injection and birds are identified that contain the active transgene in their genome, as described in Example 10. G1 birds which contain the transgene in their genome are produced from germline chimeras using methods known in the art.

Although preferred embodiments of the invention have been described using specific terms, devices, and methods, such description is for illustrative purposes only. The words used are words of description rather than of limitation. It is to be understood that changes and variations may be made by those of ordinary skill in the art without departing from the spirit or the scope of the present invention, which is set forth in the following claims. In addition, it should be understood that aspects of the various embodiments may be interchanged both in whole or in part. 

1. A method of producing a protein comprising introducing into an avian genome an exogenous nucleotide sequence comprising a fragment of the nucleotide sequence of SEQ ID NO: 67 operably linked to an exogenous coding sequence wherein the avian produces an exogenous protein.
 2. The method of claim 1 wherein the protein is deposited in egg white produced by the avian.
 3. The method of claim 1 comprising isolating the exogenous protein.
 4. The method of claim 1 wherein the exogenous protein is secreted from an oviduct cell.
 5. The method of claim 4 comprising isolating the exogenous protein.
 6. The method of claim 1 wherein the avian is selected from the group consisting of a chicken, a turkey and a quail.
 7. The method of claim 1 wherein the nucleotide sequence comprises a vector.
 8. The method of claim 7 wherein the vector is selected from the group consisting of a plasmid, a viral vector and an artificial chromosome.
 9. The method of claim 1 wherein the exogenous protein is an antibody or an enzyme.
 10. The method of claim 1 comprising isolating the exogenous protein wherein the exogenous protein is selected from the group consisting of fusion proteins, growth hormones, cytokines, structural proteins, enzymes, human growth hormone, interferon, lysozyme, and β-casein, albumin, α-1 antitrypsin, antithrombin III, collagen, factor VIII, factor IX, factor X, fibrinogen, insulin, lactoferrin, protein C, erythropoietin (EPO), granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), tissue-type plasminogen activator (tPA), somatotropin, chymotrypsin, immunoglobulins, antibodies, immunotoxins, b-domain deleted factor VIII, factor VIIa, anticoagulants; hirudin, alteplase, tpa, reteplase, tpa, tpa—3 of 5 domains deleted, insulin, insulin lispro, insulin aspart, insulin glargine, long-acting insulin analogs, glucagons, follitropin-beta, pth, fsh, pdgh, ifn alpha2, ifn alpha2a, ifn alpha2b, inf-apha, inf-beta, inf-beta 1b, ifn-beta 1a, ifn-gamma1b, il-2, il-11, hbsag, ospa, mab directed against t-lymphocyte antigen, mab directed against tag-72, tumor-associated glycoprotein, chimeric mab directed against platelet surface receptor gpII(b)/III(a), mab directed against tumor-associated antigen ca125, mab directed against human carcinoembryonic antigen, cea, mab directed against human cardiac myosin, mab directed against tumor surface antigen psma, mab directed against hmw-maa, mab directed against carcinoma-associated antigen, mab directed against nca 90, mab directed against cd20 antigen found on surface of b lymphocytes, mab directed against the alpha chain of the il2 receptor, mab directed against the alpha chain of the il2 receptor, mab directed against tnf-alpha, mab directed against an epitope on the surface of respiratory synctial virus, mab directed against her 2, human epidermal growth factor receptor 2, human mab directed against cytokeratin tumor-associated antigen anti-ctla4, mab directed against cd 20 surface antigen of b lymphocytes dornase-alpha dnase, beta glucocerebrosidase, tnf-alpha, il-2-diptheria toxin fusion protein, tnfr-lgg fusion protein laronidase, dnaases, alefacept, tositumomab, mab, alemtuzumab, rasburicase, agalsidase beta, teriparatide, parathyroid hormone derivatives, adalimumab, anakinra, biological modifier, nesiritide, human b-type natriuretic peptide (hbnp), colony stimulating factors, pegvisomant, human growth hormone receptor antagonist, protein c, omalizumab, immunoglobulin e (lge) blocker, lbritumomab tiuxetan, ACTH, glucagon, somatostatin, somatotropin, thymosin, parathyroid hormone, pigmentary hormones, somatomedin, luteinizing hormone, chorionic gonadotropin, hypothalmic releasing factors, antidiuretic hormones, prolactin and thyroid stimulating hormone.
 11. A method comprising producing a protein comprising introducing a nucleotide sequence at least 95% identical to nucleotides 7665 to 11863 of SEQ ID NO: 67 operably linked to an exogenous protein coding sequence into an oviduct cell wherein the exogenous protein is expressed in the oviduct cell.
 12. The method of claim 11 wherein the exogenous protein is secreted from the oviduct cell.
 13. The method of claim 11 wherein the avian oviduct cell is a cell of a chicken.
 14. The method of claim 11 wherein the oviduct cell is a tubular gland cell.
 15. The method of claim 11 wherein the avian lysozyme gene expression controlling region comprises a nucleotide sequence at least 99% identical to nucleotides 7665 to 11863 of SEQ ID NO:
 67. 16. The method of claim 11 wherein the avian lysozyme gene expression controlling region comprises a nucleotide sequence identical to nucleotides 5381 to 11863 of SEQ ID NO:
 67. 17. The method of claim 11 comprising isolating the exogenous protein.
 18. The method of claim 11 wherein the exogenous protein is a therapeutic protein and the therapeutic protein is isolated.
 19. A method of producing a therapeutic protein comprising introducing into an avian genome an exogenous nucleotide sequence comprising a fragment of the nucleotide sequence of SEQ ID NO: 67 wherein the avian produces a therapeutic protein in an oviduct cell and secretes the therapeutic protein from the oviduct cell.
 20. The method of claim 20 comprising isolating the therapeutic protein. 